Bloodlines of the Slave Trade examines the lives of two people whose only connection is a genetic link to John Armfield, one of the most notorious slave traders of the 1830s. Rodney Williams, who is Black, and Susanna Grannis, who is white, each trace their ancestry back to their distant ancestor, detailing the diverging paths their lineages took. While their relationship to this past is fundamentally different, and they never meet in the film, they both share in the telling of the horrific domestic slave trade and the ongoing reverberations of slavery.

The film also navigates the lesser known "second middle passage" referred to as the "domestic slave trade". Starting in Alexandria, VA, where two of the wealthiest and most infamous slave traders of the mid-19th century were headquartered, Williams journeys along the Natchez Trace where in all likelihood his ancestors walked before him. In Alexandria, John Armfield and Isaac Franklin would either ship or march the enslaved down south to Mississippi or Louisiana for both future sale and brutal work on southern plantations. These cruel transactions involved separation from family members, long and arduous journeys chained together in coffles, and even more brutal working conditions once sold off in Natchez or New Orleans. His path along the trail illuminates the mechanisms and realities of chattel slavery, and illustrates the vast accumulation of wealth created by enslaved people, but held by slave owners and benefiting their descendants.

While for a long time Susanna tried to ignore her family's history, Rodney and his siblings would not have that privilege. Tracing the ongoing impact of this shared ancestry in each of their lives, Susanna finds it to be an awakening, while for Rodney it is an exploration of the ongoing damage inflicted by the slave traders. Concluding the journey at Angola State Penitentiary, a plantation owned by Isaac Franklin, Rodney narrates his family's history wherein the link between Black incarceration and the plantation system is as explicit as it could be. Through the narratives of their families and this historical tour, Bloodlines of the Slave Trade illuminates how histories develop, become calcified, and perpetuate through generations, and posits a more honest way to discuss and understand the story of America.