Lee was taking part in the U.S. version of the genealogy documentary series 'Who Do You Think You Are?' when he made the discovery.
Lee, who made Do The Right Thing, Malcom X and She's Gotta Have It traced back his ancestry in Atlanta, Georgia, confronting a number of issues including slavery and meeting a new relative.
His first stop was at the Dublin library at the Atlanta University Center, where he met archivist Melvin Collier.
The director wanted to get information about his great great grandmother Lucinda Jackson, who was born into slavery and died in 1934.
Mr Collier helped him find a newspaper obituary for Jackson that named her
three sons but not Lucinda's husband.
He later found out that his name was Mars.
In Lee's debut movie, 'She's Gotta Have It' he named his character Mars, inspired by his grandmother, who had mentioned someone with that name who she said was a 'crazy uncle'.
Discovery: Lee investigates his slave ancestors, discovering that he could be a descendant of a slave owner, and meets Guinevere Grier, pictured, a white woman who is his cousin twice removed
The film director tells Guinevere 'I always knew who I am,' Lee tells her. 'Now I know more'
Lee noted that Mars in the movie was also crazy.
The 53-year-old director then visited the Georgia archives in Atlanta to find out more information on Mars, who he knew as a man born as a slave.
If like Spike Lee you are also interested in researching your family history, there are an increasing number of internet based resources that can help.
Websites like Genes Reunited with millions of birth, marriage and death certificates, census records and other tools that means you too can trace your family tree from the comfort of your home.
Read more about Spike Lee discovers he is mixed race...