Abstract Background: Colombia reports one of the highest numbers of endemic Leishmania species (n=9) causing human disease . Although much effort has been devoted to generating simple and specific tools for Leishmania species identification, challenges remain in the discrimination of species belonging to the Leishmania (Viannia) guyanensis complex: L. (V.) guyanensis and L. (V.) panamensis . Methods: A set of seven reference strains of species pertaining to the L. Leishmania and L. Viannia subgenera, clinical strains from human cases of cutaneous leishmaniasis (CL, n=26), and samples collected from sylvatic mammals and sandflies (n=7) from endemic areas in Colombia were included in this study. The hsp70 gene was PCR-amplified from DNA extracts of logarithmic phase promastigotes or tissue samples, and PCR products were sequenced. Sequence alignment was performed against a set of previously published and curated sequences, and phylogenetic analysis based on maximum-likelihood and Bayesian inference approaches was conducted. Haplotypic diversity among strains and species of the L.(V.) guyanensis complex was explored using a median-joining network. Results: Sequencing of the hsp70 gene for L.Viannia species typing was comparable to species identification using isoenzyme electrophoresis or monoclonal antibodies. Complete species matching was found, except for one sylvatic sample with an identity yet unsolved. Among the L. (V.) panamensis clinical strains , we found two distinctive phylogenetic clusters correlated with two different zymodemes, L.(V.) panamensis Z2.2 and Z2.3. Analysis of samples from sylvatic environments identified novel records of wild mammals and sandflies species naturally infected. Conclusions: Our results support the adequacy of hsp70 gene sequencing as a single locus approach for discrimination of L.Viannia species, as well as for exploring the genetic diversity within the L. (V.) guyanensis complex.