Leishmaniasis is a disease caused by parasites of the genusLeishmaniaand transmitted by sand fly vectors. Tegumentary leishmaniasis is the most prevalent clinical outcome in Latin America, afflicting people from 18 countries. In Panama, the annual incidence rate of leishmaniasis is as high as 3000 cases, representing a major public health problem. In endemic regions,L. panamensisis responsible for almost eighty percent of human cases that present different clinical outcomes. These differences in disease outcomes could be the result of the local interplay betweenL. panamensisvariants and human hosts with different genetic backgrounds. The genetic diversity ofL. panamensisin Panama has only been partially explored, and the variability reported for this species is based on few studies restricted to small populations and/or with poor resolutive markers at low taxonomic levels. Accordingly, in this study, we explored the genetic diversity of sixty-nineL. panamensisisolates from different endemic regions of Panama, using an MLST approach based on four housekeeping genes (Aconitase,ALAT,GPIandHSP70). Two to seven haplotypes per locus were identified, and regional differences in the genetic diversity ofL. panamensiswere observed. A genotype analysis evidenced the circulation of thirteenL. panamensisgenotypes, a fact that might have important implications for the local control of the disease.