Genome plasticity has been hypothesized to be a driving force behind parasite speciation. We isolated 17 low to moderate copy genes and evaluated the divergence of such genes in terms of locus organization chromosomal location and gene expression in old world Leishmanias (OW). Analysis was performed to identify restrictionlength polymorphisms (RFLPs) providing heritable markers for OW leishmanias. RFLP markers were conserved in parasite isolates from primary infections demonstrating their utility as diagnostic tools. The 17 genes were distributed among 9 distinct chromosomes and each gene was localized to a similar sized chromosome from each isolate. Molecular tree derived from PFLP data suggest that OW leishmanias comprise a monophyletic lineage, with species associated with cutaneous disease exhibiting the greatest level of divergence. Further, species causing cutaneous and visceral disease have diverged primarily by nucleotide substitutions.