Chambers was born in Springfield, Ohio, the son of Pam and John Chambers. He has a fraternal twin brother named Jason, one older brother John Jr. (Chip), and two older sisters, Mia and Susan. He and his twin brother visited hospitals often as children due to frequent bouts with pneumonia. He attended Southeastern High School in South Charleston, Ohio, and later Springfield Clark JVS.
Chambers was discovered by a modeling scout on the Paris Metro and went on to represent Calvin Klein in a fragrance campaign. He worked throughout Europe, Japan, and the United States for Calvin Klein, Armani, and Dolce & Gabbana. After deciding on an acting career, he moved to New York, where he studied at the HB Studio. Studios and Ron Stetson Studios for four years. That landed him a few television roles, including recurring role in soap opera Another World as Nicholas Hudson, and New York Undercover. In 1998 he was cast as series regular in the short-lived CBS primetime soap opera Four Corners. Chambers also appeared in several television movies, such as Harvest of Fire, Rose Hill, and Seasons of Love
Chambers starred in several movies, notably Liberty Heights (1999, as Trey, alongside Adrien Brody),The Wedding Planner (2001, as Massimo, alongside Jennifer Lopez), The Musketeer (2001) Southern Belles (2005), The Zodiac (2005), and the HBO original movie Hysterical Blindness (2002). In 2003, Chambers was cast as one of the leads in the CBS crime drama Cold Case but left after appearing in only four episodes.
In August, 2004, Chambers was cast as surgical intern Alex Karev in Shonda Rhimes medical drama series Grey's Anatomy, opposite Ellen Pompeo, Sandra Oh, Katherine Heigl, and T. R. Knight. In 2006, along with other cast members, he was awarded a Screen Actors Guild Award for Outstanding Performance by an Ensemble in a Drama Series. In 2009, he also guest-starred on the Grey's Anatomy spin-off, Private Practice, for the Grey's Anatomy/Private Practice crossover event.
In 1993, he married Keisha, a former model agency booker. They have five children together: Isabella (b. December 1994), twins Maya and Kaila (b. June 1997), Eva (b. March 1999) and Jackson (b. January 2002). His wife and children have recently moved from their home in a suburb just outside of New York City to Los Angeles.