As shown by RFLP analysis, there is a high variability in the β-tubulin gene region of Leishmania sp. Such variability has been used in the identification of these parasites, establishing differences between subgenera of New World Leishmania . We have found a region of 500 bp (β500) upstream of the coding region of the β-tubulin gene that is present in all strains tested belonging to the L. (Viannia) subgenus. This region apparently is a repetitive sequence and we have shown that it is specific to the Leishmania (Viannia) subgenus. This sequence has no homology with the genomic DNA isolated from either the species belonging to the L. (Leishmania) subgenus or other Kinetoplastida, such as Trypanosoma cruzi , T. brucei , Leptomonas samueli , or Crithidia fasciculata . The β500 sequence showed sufficient variation to be used as a molecular marker in the identification of parasites. We established inter- and intrasubgenus differentiation and were able to discriminate at the species level in the Vianna subgenus. A PCR assay confirmed the specificity of the β500 sequence.