It has been long appreciated that speciation involves changes in body plans and establishes genetic, reproductive, developmental and behavioral incompatibilities between populations. However, little is still known about the genetic components involved in these changes or the sequence and scale of events that lead to the differentiation of species.In this paper, we investigated the genetic changes in three closely related species of Drosophila by making pair-wise comparisons of their genomes. We focused our analysis on the modern relatives of the alleles likely to be segregating in pre-historic populations at the time or after the ancestor of D. simulans became separated from the ancestor of D. melanogaster. Some of these genes were previously implicated in the genetics of reproduction and behavior while the biological functions of others are not yet clear.Together these results identify different classes of genes that might have participated in the beginning of segregation of these species millions of years ago in Africa.