We have previously identified a novel genomic sequence of 500 bp, the beta 500-DNA sequence, in the subgenus Leishmania (Viannia). This sequence was localized upstream of the beta-tubulin gene. Restriction fragment length polymorphism and hybridization analysis has shown that the beta 500-DNA sequence is specific to this subgenus. A polymerase chain reaction (PCR) assay confirmed this specificity. The beta 500-DNA sequence was apparently absent from the genomic deoxyribonucleic acid of L. colombiensis and L. equatoriensis. These results indicate that a PCR assay based on the beta 500-DNA sequence is likely to be of use to detect and identify Leishmania parasites of this subgenus in clinical samples with high sensitivity, specificity and reliability. The beta 500-DNA sequence can be considered a molecular marker for the subgenus Viannia.