Abstract Leishmaniasis is a disease representing an important public health problem worldwide, with a broad spectrum of clinical and epidemiological features partly associated with the diversity and complex life cycle of the Leishmania parasites. This study analyzes genomic data from 205 Leishmania (Viannia) samples, including 66 newly sequenced clinical isolates. It also provides chromosome-level genome assemblies for 10 isolates representing different species and populations. The observed distribution of Leishmania genomic diversity across the sampling locations suggests rapid adaptation to different ecosystems. Pangenomic analysis of high-quality assemblies shows consistent copy number variation between species for different gene families. Amastin gene families have larger numbers and diversity than previous reports based on analysis of short-read data. This work provides comprehensive genomic resources to identify population markers for Leishmania spp, leveraging valuable insights into the biology, transmission dynamics, the evolution of virulence mechanisms, and the spread of resistance of the parasite.