In the Americas, the sandfly Lutzomyia longipalpis is the main vector of the parasitic protozoa Leishmania infantum, the etiological agent of visceral leishmaniasis (VL). The Lu. longipalpis species complex is currently discontinuously distributed across the Neotropical region, from Mexico to the north of Argentina and Uruguay. During its continental spreading, it must have adapted to several biomes and temperature amplitudes, when founder events should have contributed to the high genetic divergence and geographical structure currently observed, reinforcing the speciation process. The first report of Lu. longipalpis in Uruguay was in 2010, calling the attention of Public Health authorities. Five years later, the parasite Le. infantum was recorded and in 2015 the first case of VL in canids was reported. Hitherto seven human deaths by VL have been reported in Uruguay. Here, we publish the first DNA sequences from the mitochondrial genes ND4 and CYTB of Lu. longipalpis collected in Uruguay, and we used these molecular markers to investigate their genetic variability and population structure. We described four new ND4 haplotypes in a total of 98 (4/98) and one CYTB in a total of 77 (1/77). As expected, we were able to establish that the Lu. longipalpis collected in two localities (i.e. Salto and Bella Unión) from the north of Uruguay are closely related to the populations from neighbouring countries. We also propose that the possible route for the vector arrival to the region may have been through vegetation and forest corridors of the Uruguay River system, as well as it may have benefited from landscape modifications generated by commercial forestation. The ecological-scale processes shaping Lu. longipalpis populations, the identification of genetically homogeneous groups and the gene flow among them must be carefully investigated by using highly sensible molecular markers (i.e. genome wide SNPs) since it will help to the understanding of VL transmission and contribute to the planification of public policies on its control. © 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.