A strand of reddish blond hair discovered inside a Bible once owned by Thomas Jefferson sparked a genetic investigation that lasted more than a decade. The findings now provide the strongest evidence to date connecting Jefferson to descendants of Sally Hemings, an enslaved woman in his household.

Staff at the Smithsonian National Museum of American History found the hair pressed between the Bible’s pages in 2011. Researchers initially suspected it had belonged to Jefferson, offering a possible source of his DNA.

Scientists ultimately reconstructed DNA fragments and compared genetic material from several family lines. Their analysis indicates a 99.96% chance that Jefferson fathered at least one of Hemings’ children.

The investigation is documented in “History by a Hair: Thomas Jefferson, Sally Hemings & Solving a 200-Year-Old Mystery,” written by Richard Kurin, a distinguished scholar and ambassador at large at the Smithsonian. The book was published on September 22.

Kurin is also senior author of a paper detailing the genetic methods used to connect the former United States president with Hemings’ descendants. The paper was published September 21 on the preprint platform bioRXiv and has not yet undergone peer review.

Researchers examined the Bible hair and genetic material from confirmed Jefferson hair samples held by the Smithsonian. They compared the resulting data with DNA from descendants of Jefferson and his wife Martha, Jefferson’s brother Randolph, and Hemings’ sons Eston and Madison.

Hemings had four children who survived to adulthood, but only Eston and Madison identified Jefferson as their father. The researchers collected DNA from multiple descendants of both men while examining patterns across the Jefferson and Hemings family trees.

A 1998 genetic study led by Eugene Foster, a retired pathology professor at Tufts University and the University of Virginia, previously claimed evidence of Jefferson’s parentage. It identified a rare haplotype in the Y chromosomes of men descended from Jefferson’s uncle and in Hemings’ descendants.

Critics said that evidence connected Hemings’ descendants to a man in the broader Jefferson lineage, not necessarily Thomas Jefferson. “He didn’t have Thomas Jefferson’s DNA,” Kurin said.

Kurin believed obtaining Jefferson’s DNA could provide a direct point of comparison with Hemings’ descendants. “I thought, if we can get Thomas Jefferson’s DNA, we could use that as the source of comparison to various descendants, and really firmly solve the question,” he said.

The hair from the Bible did not provide that breakthrough. Researchers compared its mitochondrial DNA with DNA from a living woman descended directly from Jefferson’s mother through the family’s female lineage, but the samples did not match.

The team then obtained other hair samples that historical records attributed to Jefferson, including a lock clipped at his deathbed. The hairs were stored in a packet provided by the Thomas Jefferson Foundation at Monticello and had not previously been available for analysis.

Scientists reconstructed and compared historic genome sequences of Jefferson and Hemings with DNA collected from 16 living people. The participants included multiple descendants of Eston and Madison, and the analysis found genome wide matches connecting Hemings’ descendants to Jefferson.

“What made this study so powerful is that researchers weren’t just comparing two genomes,” said Éadaoin Harney, a senior population geneticist at the 23andMe Research Institute who was not involved in the research. “Instead, they collected DNA samples from multiple people from different branches of the family tree.”

Questions about Jefferson’s sexual relationship with Hemings, who was 30 years younger and a half sister to his wife, have persisted for more than 200 years. Records aligning Hemings’ pregnancies with Jefferson’s presence at Monticello helped sustain those accounts.

In 2000, the Thomas Jefferson Foundation acknowledged “the strong likelihood that Thomas Jefferson and Sally Hemings had a relationship over time that led to the birth of one, and perhaps all, of the known children of Sally Hemings.” Its statement followed the disputed 1998 genetic findings.

The Bible hair still holds an unresolved mystery. Researchers determined from its fragmentary genetic data that it belonged to a female member of the Jefferson family, but they could not identify her.

“It’s like a partial fingerprint,” Kurin said. “That is still a lingering mystery.”