The genetic split in Africa resulted in distinct populations that lived in isolation for as much as 100,000 years, the scientists say.
This could have been caused by arid conditions driving a wedge between humans in eastern and southern Africa.
Details have been published in the American Journal of Human Genetics.
It would be the longest period for which modern human populations have been isolated from one another. But other scientists said it was still too early to reconstruct a meaningful picture of humankind's early history in Africa. They argue that other scenarios could also account for the data. At the time of the split - some 150,000 years ago - our species, Homo sapiens, was still confined to the African continent.
The results have come from the Genographic Project, a major effort to track human migrations through DNA. The latest conclusions are based on analysis of mitochondrial DNA in present-day African populations. This type of DNA is the genetic material stored in mitochondria - the "powerhouses" of cells. It is passed down from a mother to her offspring, providing a unique record of maternal inheritance.
"We don't know how long it takes for hominids to fission off into separate species, but clearly they were separated for a very long time," said Dr Spencer Wells, director of the Genographic Project. "They came back together again during the Late Stone Age - driven by population expansion."
Although present-day people carry a signature of the ancient split in their DNA, today's Africans are part of a single population. The researchers compiled a "family tree" of different mitochondrial DNA groupings found in Africa. A major split occurred near the root of the tree as early as 150,000 years ago. On one side of this divide are the mitochondrial lineages now found predominantly in East and West Africa, and all maternal lineages found outside Africa.
On the other side of the divide are lineages predominantly found in the Khoi and San (Khoisan) hunter-gatherer people of southern Africa. Many African populations today harbour a mixture of both. The scientists say the most likely scenario is that two populations went their separate ways early in our evolutionary history.
This gave rise to separate human communities localised to eastern and southern Africa that evolved in isolation for between 50,000 and 100,000 years. This divergence could have been related to climate change: recent studies of ancient climate data suggest that eastern Africa went through a series of massive droughts between 135,000-90,000 years ago.
Lead author Doron Behar, from the Rambam Medical Center in Israel commented: "It is possible the harsh environment and changing climate made populations migrate to other places in order to have a better chance of survival. "Some of them found places where they could and - perhaps - some didn't. More than that we cannot say."
Dr Wells told BBC News: "Once this population reached southern Africa, it was cut off from the eastern African population by these drought events which were on the route between them." Modern humans are often presumed to have originated in East Africa and then spread out to populate other areas. But the data could equally support an origin in southern Africa followed by a migration to East and West Africa.
The genetic data show that populations came back together as a single, pan-African population about 40,000 years ago. This renewed contact appears to coincide with the development of more advanced stone tool technology and may have been helped by more favourable environmental conditions.
"[The mixing] was two-way to a certain extent, but the majority of mitochondrial lineages seem to have come from north-eastern Africa down to the south," said Spencer Wells.
But other scientists said different scenarios could explain the data. Dr Sarah Tishkoff, an expert on African population genetics from the University of Pennsylvania, said the Khoisan might once have carried many more of the presumed "East African" lineages but that these could have been lost over time.
"Although there is very deep divergence in the mitochondrial lineages, that can be different from inferring when the populations diverged from one another and there can be many demographic scenarios to account for it," she told BBC News. She added: "As a general rule of thumb, when mitochondrial genetic lineages split, it will usually precede the population split. It can often be difficult to infer from one to the other."
The University of Pennsylvania researcher stressed it was not possible to pinpoint where in Africa the populations had once lived - complicating the process of reconstructing scenarios from genetic data. The Genographic Project's findings are also consistent with the idea - held for some years now - that modern humans had a close brush with extinction in the evolutionary past. The number of early humans may have shrunk as low as 2,000 before numbers began to expand again in the Late Stone Age. - bbc