Visceral leishmaniasis (VL) is a life-threatening disease caused by the protozoa Leishmania donovani and L. infantum. Likely, L. infantum was introduced in the New World by the Iberic colonizers. Due to recent introduction, the genetic diversity is low. Access to genomic information through the sequencing of Leishmania isolates allows the characterization of populations through the identification and analysis of variations. Population structure information may reveal important data on disease dynamics. Aiming to describe the genetic diversity of L. infantum from the Middle-North, Brazil, next generation sequencing of 30 Leishmania isolates obtained in the city of Teresina, from where the disease dispersed, was performed. The variations were categorized accordingly to the genome region and impact and provided the basis for chromosomal ploidy and population structure analysis. The results showed low diversity between the isolates and the Iberic reference genome JPCM5. Most variations were seen in non-coding regions, with modifying impact. The ploidy number analysis showed aneuploid profile. The population structure analysis revealed the presence of two L. infantum populations identified in Teresina. Further population genetics studies with a larger number of isolates should be performed in order to identify the genetic background associated with virulence and parasite ecology.