Sand fly species are traditionally identified using morphological traits, though this method is hampered by the presence of cryptic species. DNA barcoding is a widely used tool in the case of insects of medical importance, where it is necessary to know quickly which species are present in a transmission area. Here, we assess the usefulness of mitochondrial cytochromecoxidase subunit I (COI) DNA barcoding as a practical tool for species identification, correct assignment of isomorphic females, and to evaluate the detection of cryptic diversity that occurs in the same species. A fragment of theCOIgene was used to generate 156 new barcode sequences for sand flies from different countries of the Neotropical region, mainly Colombia, which had been identified morphologically as 43 species. The sequencing of theCOIgene allowed the detection of cryptic diversity within species and correctly associated isomorphic females with males identified by morphology. The maximum intraspecific genetic distances ranged from 0 to 8.32% and 0 to 8.92% using uncorrectedpdistances and the Kimura 2-parameter (K2P) model, respectively. The minimum interspecific distance (nearest neighbor) for each species ranged from 1.5 to 14.14% and 1.51 to 15.7% usingpand K2P distances, respectively. Three species had more than 3% maximum intraspecific distance:Psychodopygus panamensis,Micropygomyia cayennensis cayennensis, andPintomyia evansi. They also were split into at least two molecular operational taxonomic units (MOTUs) each, using different species delimitation algorithms. Regarding interspecific genetic distances, the species of the generaNyssomyiaandTrichophoromyiagenerated values lower than 3% (exceptNyssomyia ylephiletorandNy. trapidoi). However, the maximum intraspecific distances did not exceed these values, indicating the presence of a barcode gap despite their proximity. Also, nine sand fly species were DNA barcoded for the first time:Evandromyia georgii,Lutzomyia sherlocki,Ny. ylephiletor,Ny. yuilli pajoti,Psathyromyia punctigeniculata,Sciopemyia preclara,Trichopygomyia triramula,Trichophoromyia howardi, andTh. velezbernali. TheCOIDNA barcode analysis enabled the correct delimitation of several Neotropical sand fly species from South and Central America and raised questions about the presence of cryptic species for some taxa, which should be further assessed.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05807-z.