Groff graduated from Conestoga Valley High School in 2003 and intended to attend Carnegie Mellon University, but deferred his admission for a year when he was cast as Rolf in a Non-Equity national tour of The Sound of Music. After the tour, Groff decided to move to New York City instead. While still living in Lancaster, Groff performed at the Fulton Opera House in The Sound of Music, Ragtime, Evita, My Fair Lady, Peter Pan, The Pirates of Penzance, and Rags and The Ephrata Performing Arts Center as Edgar in Bat Boy: The Musical and Ugly in Honk!.

Groff earned his Actors’ Equity Association card in 2005, with the musical Fame at the North Shore Music Theatre. That same year, he made his Broadway debut as an understudy for the lead role and a swing for the musical In My Life by Joseph Brooks. Groff originated the role of Melchior Gabor in the Broadway production of the rock musical Spring Awakening. He played the role from its debut on December 10, 2006, through May 18, 2008, when he departed the production with his co-star and best friend Lea Michele. He had previously played the role in the original Off-Broadway production earlier in the summer of 2006. Groff was nominated for a Drama Desk Award and a Tony Award for Best Leading Actor in a Musical for his performance.

He played the recurring role of Henry Mackler on the ABC soap opera One Life to Live. His storyline about a school shooting was cut due to the Virginia Tech shooting in April 2007, and Groff only appeared in eleven episodes. From July 22 through August 31, 2008, Groff played as Claude in the Shakespeare in the Park production of Hair. He also appeared as Michael Lang in Ang Lee’s comedy-drama film Taking Woodstock. Groff next appeared in the off-Broadway production of the play Prayer for My Enemy by Craig Lucas. In August 2009, Groff performed in The Bacchae as Dionysus, as a part of the Public Theater’s Shakespeare in the Park.

Groff publicly came out as gay during the National Equality March in 2009 and was romantically linked to actor Gavin Creel that same year. From 2010 to 2013, he was in a relationship with actor Zachary Quinto. Since early 2018, he has been dating New Zealand choreographer Corey Baker, whom he met while both were teaching at Christchurch International Musical Theatre Summer School (CIMTSS). Groff was diagnosed with melanoma in his early 20s.

In 2010, he guest-starred in eight episodes of the Fox musical comedy-drama Glee as Jesse St. James alongside Lea Michele. Groff continued to guest-star as the character in subsequent seasons, including the final episode of the series. In August 2010, he made his West End debut in Deathtrap, at the Noël Coward Theatre, directed by Matthew Warchus.

From August to October 2012, Groff appeared as Ian Todd in the second and final season of the Starz series Boss. Groff then portrayed Ken in the Center Theatre Group’s production of the play Red, alongside Alfred Molina. The show ran from August 1 through September 9, 2012. In March 2013, Groff and Molina reprised their roles for six more performances of the play, this time in the L.A. Theatre Works. These performances were recorded for broadcast on radio.

In 2013, Groff voiced one of the lead roles in Disney’s animated feature Frozen. His character, Kristoff, is a rugged mountain man and ice trader. The film premiered on November 19, 2013. Following the film’s massive commercial success and its ranking as the highest-grossing animated film of all time, the fifteenth highest-grossing film of all time, and the highest-grossing film of 2013, Groff reprised his role in the short film sequel Frozen Fever, which premiered on March 13, 2015.

Groff starred as Patrick Murray, a gay video game developer, in HBO’s comedy-drama series Looking. On March 23, 2015, HBO cancelled Looking after its second season but greenlit a television film to conclude the story. In April 2013, Groff joined another HBO production, playing Craig Donner in the film adaptation of Larry Kramer’s play The Normal Heart. He returned to the London stage on May 19, 2015, to star in a one-night-only concert of the musical How to Succeed in Business Without Really Trying, at the Royal Festival Hall.

Groff starred in a production of the musical A New Brain, presented as part of the New York City Center’s Encores! Off-Center staged concert series. The show was directed by James Lapine and took place from June 24 through June 27, 2015. On March 3, 2015, he joined the cast of the musical Hamilton, replacing Brian d’Arcy James in the role of King George III. He held the role for the remainder of the show’s off-Broadway production, through May 3, 2015. He also appeared in as King George III in Hamilton which was added to Disney+ on July 3, 2020. He reprised the role in the Broadway production, which started performances July 13, 2015. Groff, along with the rest of the cast, won a Grammy Award for Best Musical Theater Album for appearing as a featured performer on the original Broadway cast recording. He was also nominated for the Tony Award for Best Featured Actor in a Musical.

In April 2015, Groff was honored by the Point Foundation with the Point Horizon Award honoring a “trailblazer who has taken a leadership role as an advocate” of LGBTQ communities. In December 2015, he was honored by Equality Pennsylvania with the Bayard Rustin Award, which “recognizes a Pennsylvanian who is continuing the work to ensure that the LGBT community will be visible, accepted, and celebrated in our society.”

In March 2016, it was reported that Groff would star in the Netflix series Mindhunter, executive produced by David Fincher and Charlize Theron. The series was released in October 2017, and he portrays Federal Bureau of Investigation agent Holden Ford, a serial killer investigator in the Behavioral Science Unit. Groff also starred in the second season of the series, which premiered on August 16, 2019.

In July 2017, Groff starred in the first podcast musical, 36 Questions, which was released in three parts.

In November 2019, Groff reprised his role as Kristoff in Frozen II. He sang a solo song for the movie, titled “Lost in the Woods”.