SUMMARYWe used 12 microsatellite markers developed forLeishmania braziliensisto genotype 28 strains of the main species of theLeishmania guyanensiscomplex (i.e. L. guyanensisandL. panamensis) collected in Ecuador and Peru. The important heterozygote deficits observed in these populations are similar with the previous data obtained inL. braziliensisand raise again the debate on the reproductive mode of these protozoan parasites. The data showed genetic polymorphism and geographical differentiation giving information on population structure of theL. guyanensiscomplex. Regarding the two species, this study enhances again the debate on the taxonomic status of the different isolates belonging toL. guyanensiss.l. since the results showed substantial heterogeneity within this species complex. In conclusion, this study increases the number of available microsatellite loci forL. guyanensisspecies complex and raises fundamental biological questions. It confirms that microsatellite markers constitute good tools for population genetic studies on parasites of this complex.