Published in the Proceedings of the National Academy of Sciences, the study indicated that a mild and more severe strain diverged either 16,000 or 68,000 years before present, depending on whether accounts from East Asia or Africa are used to calibrate the molecular clock.
The researchers claim that in either case, the divergence stretches further back in time than previously believed.
Vaccinations for smallpox were stopped in 1972, and the disease was considered eradicated in 1980, three years after the last naturally occurring case in 1977.
The researchers said that the correlation of historical records and a molecular clock paves the way for them to study the natural history of other history.
They reckon that the human disease might have originated from a rodent-borne virus in Africa.
Upon evolutionary analysis, the researchers came to the conclusion that smallpox slowly spread westward from East Asia, which would agree with the oldest smallpox-like descriptions from ancient China as far back as 1122 BC.
The researchers believe that the slow spread out of Asia may help understand why smallpox descriptions are missing from ancient Greece or Rome as well as the Old and New Testaments.
Researchers Shea Gardner and Yu Li have devised a way to concentrate point mutations in the viral DNA, single nucleotide polymorphisms, or "SNPs".
They looked across the nearly 200,000 DNA base pairs of the virus genome, and concentrated the mutations for comparing sequences by excerpting stretches in which a single change at one point was flanked by seven unchanged bases at each side.
"We assumed there was a molecular clock ticking. The question was what was the rate?" Gardner said.
Li said that the Laboratory's intensive computing capabilities complemented the CDC contribution of calibrating the information with historical accounts.
"It was a valuable opportunity to be able to compare the genomes," said Lab scientist Beth Vitalis, who helped analyze the data. She added that additional, related studies of virulence factors are in process.