Burton was born in Sterling, Virginia. Her father is a former Green Beret and her mother is a real estate agent. She is the eldest of four children with three brothers. She graduated from Park View High School in 2000, where she was student council treasurer her second year, vice-president her junior year, and captain of the cheerleading squad, student council president, and homecoming queen her senior year. Burton attended New York University and Fordham University.
Burton's first big break came when working as a VJ for the Total Request Live on MTV. She was supposed to be a guest commentator for one segment, but producers decided to offer her a permanent job. Burton went on to present at the 2000 MTV Video Music Awards and MTV's Iced Out New Year's Eve. Burton went on to portray herself on The WB drama Dawson's Creek in 2002, which was marked as her television debut.
In April 2003, Burton was cast in The WB drama One Tree Hill. Burton portrayed the role of Peyton Sawyer, a fiercely independent visual and musical artist and cheerleader. The series premiered on September 23, 2003 and went on to be the network's top rated program of the year. The role was considered Burton's breakout role and is her best-known to date. For her role in the series, Burton was featured on the cover of Maxim, American Cheerleader Magazine and People. Her performance received critical praise and she earned three Teen Choice Award nominations. In May 2009, The CW announced Burton would not be returning for the show's seventh season based on her own decision not to return, contrary to rumors she left due to salary issues.
In June 2005, Burton made her feature film debut opposite Allison Janney and Cheryl Hines in the drama Our Very Own that centered on five small-town teenagers who dream of a better life and was released to positive reviews from critics. Burton and her co-stars received the "Outstanding Ensemble Acting" award at the prestigious Sarasota Film Festival. In September 2007, Burton appeared in the Lifetime drama Normal Adolescent Behavior which followed a group of friends who are in a six-way polyfidelitous relationship. Burton played Ryan. That same year Burton co-starred in the supernatural horror film Solstice opposite Amanda Seyfried and Elisabeth Harnois.
In June 2008, Burton appeared in the thriller The List as Jo Johnston and was filmed in Wilmington, North Carolina while Burton was on production break for One Tree Hill. The film had a limited release and went on to make $132,863 in the US alone. That same year, Burton appeared in the Fox Searchlight Pictures drama The Secret Life of Bees as Deborah Owens, the deceased mother of Dakota Fanning's character.
In January 2009, Burton was cast in the drama Bloodworth opposite Hilary Duff and Val Kilmer. The film is an adaptation of the novel Provinces of Night by William Gay. Filming also took place in Wilmington, North Carolina in April and May that same year. The film premiered at the 2010 Santa Barbara International Film Festival in February 2010. The film had a limited release on May 20, 2011 to positive reviews from critics and over $12,971 worldwide.
In July 2010, Variety announced Burton would have a recurring arc in the USA Network crime drama series White Collar as Sara Ellis, an insurance investigator in the second season. In February 2011, Burton was upgraded to a series regular for the third season which premiered on June 7, 2011. Burton was switched back to the recurring cast in the fourth season.
On January 23, 2012, Burton guest-starred on the ABC police comedy-drama Castle, in season 4: episode 13 titled "An Embarrassment of Bitches" as Kay Cappuccio, a reality television star who is accused of the murder of a famous dog trainer. In May 2013, Burton had a recurring role in the ABC medical drama Grey's Anatomy for the final three episodes of the ninth season, as Dr. Lauren Boswell.
In August 2013, Burton had a recurring role on the short-lived CBS drama Hostages as Samantha, the mistress of Brian Sanders.
In July 2015, Burton had a recurring role on CBS sci-fi drama Extant as Anna Schaefer where she reunited with One Tree Hill co-star Tyler Hilton.
During her time on One Tree Hill, Burton resided in Wilmington, North Carolina, where the show was filmed. In 2004, Burton married One Tree Hill assistant director Ian Prange, son of executive producer Greg Prange, but they divorced in 2009.
In May 2007, Burton ranked #77 in Maxim's "Hot 100 List of 2007". She also appeared on the cover of the November 2006 edition of Maxim with One Tree Hill co-star Sophia Bush. In previous years, Burton ranked #2 on Femme Fatales' "The 50 Sexiest Women of 2005", and #12 on Much Music's "20 Hottest Women of 2003".
In 2007, Burton created her own production company called Southern Gothic Production (SoGoPro) along with Nick Gray, Kelly Tenney, James Burton and Meg Mortimer. Burton is also on the North Carolina Film Council.
Burton began a relationship with actor Jeffrey Dean Morgan in 2009. In March 2010, she gave birth to their son Augustus ("Gus"). In a July 2015 interview with The Philippine Daily Inquirer, Morgan said that despite media reports to the contrary, he and Burton were not yet married but that they intended to wed when he could get enough time off from work The Road Mix: Music from the Television Series One Tree Hill, Volume 3 (2007)