Previous studies indicated that two distinct chromosomal forms of Anopheles nuneztovari Gabaldón, cytotypes B and C, occurred on the west and east of the Latin American Andes Mountains, respectively. To determine the taxonomic status of Anopheles (Nyssorhynchus) nuneztovari in Colombia, link-reared specimens were collected from four sites: in the departments of Chocó (La Pacurita) and Valle (Sitronella) in the west, and Norte de Santander (Guaramito and Tibú) in the east. Nuclear ITS2 sequences were generated for 46 individuals. Only two specimens (4.4%) showed divergent haplotypes, varying from the consensus by a single-base polymorphism (0.18%). These results suggest that populations of An. nuneztovari corresponding to cytotypes (B and C) are conspecific.