Martindale was born July 18, 1951 in Jacksonville, Texas, the youngest of three children and only daughter of william Everett and Margaret (Pruitt) Martindale. In addition to owning and operating a lumber company in Jacksonville, her father was known as a champion dog handler in Texas and throughout the southern United States.
Following graduation from Jacksonville High School in 1969, Martindale attended Lon Morris College, then transferred to the University of Michigan at Ann Arbor. While at Michigan, she did summer study at Harvard University, appearing onstage with Future movie and TV stars Jonathan Frakes and Christopher Reeve.
Margo Martindale has been married to musician Bill Boals since 1986. They have a daughter, Margaret.
Martindale made her Broadway debut in 2004 as Big Mama in Cat on a Hot Tin Roof. She received a Tony Award nomination for Best Featured Actress in a Play, for her work in the role.
From 2007-08, she had a recurring role as Nina Burns, a neighbor of the Malloy/"Rich" family in The Riches with Minnie Driver and Eddie Izzard.
In 2011, Martindale joined the cast of Justified for the second season. She played the role of Mags Bennett, matriarch of the Bennett crime family which controlled much of the drug activity in the fictional version of Harlan County, Kentucky. She won the Primetime Emmy Award for Outstanding Supporting Actress in a Drama Series for her performance. After learning of the nomination, Martindale told CNN she hoped that it would open up more doors for older women in Hollywood. "People really identify with this character [Mags Bennett] and I think it's because it is a character that is powerful and older and extremely mean", she said.
In February 2012 it was announced Martindale had been cast in the ABC comedy pilot Counter Culture, which was not picked up.
Martindale returned to television in late January 2013 in the spy drama The Americans on FX Network. She plays Claudia, the KGB "handler" of two Soviet spies living in 1980s Cold War America.
Martindale played Audrey Bernhardt, matriarch of the family on the Amazon series Sneaky Pete starring Giovanni Ribisi, for the 2015 pilot the first season which aired in January 2017, and the second season as well.