Different approaches are being developed to improve the differentiation of Leishmania genus using biochemical and molecular methods. In this study, 11 independent polymorphic microsatellites were used for the typing of strains of L. infantum isolated in Sicily. Polymerase chain reaction was employed to amplify the microsatellites contained in 12 DNA regions selected from among more investigated loci. A total of 51 isolates of L. infantum from dogs were tested by using the same locus panel. The products were successively analysed using an automatic sequence detector (ABI PRISM 3130 AB), to discover relevant microsatellite polymorphisms. It was possible to discriminate between MON-1 and non-MON-1 groups. Moreover, the method permitted to distinguish various genotypes of L. infantum isolates within each zymodema. Modeland distance-based analyses of the data set showed comparable results. The frequency of heterozygosity in the alleles analysed varied extremely between the different groups of isolates. As the method exhibits a high level of discrimination, it is suitable for characterization of closely related strains in epidemiological studies.