Twelve microsatellite loci of Leishmania braziliensis were examined, nine of which were developed in this work. Fifty-six Leishmania braziliensis were genotyped with these microsatellite loci. The 12 loci studied were polymorphic with the number of alleles ranging from five to 19, with a mean of 9.7 ± 4.1 and the observed heterozygosity averaging 0.425 ± 0.202. The important heterozygote deficits we observed (FIS = 0.41, P value = 0.004) appear incompatible with the heterozygote excess expected in clonal diploids. This last result could revive the clonality/sexuality debate regarding Leishmania. This work validates the potential use of these microsatellites for population genetics analysis. © 2007 The Authors.