The subfamily Phlebotominae comprises important insects for public health. The use of complementary tools such as molecular taxonomy is necessary for interspecific delimitation and/or discovery of cryptic species. Here, we evaluated the DNA barcoding tool to identify different species in the southwestern Brazilian Amazon. For this, we collected sand flies in forest fragments along the highway BR-317, in the municipality of Brasiléia, state of Acre, Brazil. The specimens were DNA-barcoded using a fragment of the cytochrome c oxidase subunit I (COI) gene. The sequences were analyzed to generate K2P pairwise genetic distances and a Neighbour-joining tree. The sand fly barcodes were also clustered into Molecular Operation Taxonomic Units (MOTU) using Automatic Barcode Gap Discovery (ABGD) approach. A total of 59 COI sequences comprising 22 nominal species and ten genera were generated. Of these, 11 species had not been sequenced before, thus being new COI sequences to science. Intraspecific genetic distances ranged between 0 and 4.9%, with Pintomyia serrana presenting the highest values of genetic distance, in addition to having been partitioned into three MOTUs. Regarding the distances to the nearest neighbour, all species present higher values in relation to the maximum intraspecific distance, in addition to forming well supported clusters in the neighbour-joining analysis. The DNA barcoding approach is useful for the molecular identification of sand flies from Brasiléia, state of Acre, and was efficient in detecting cryptic diversity of five species which can be confirmed in future studies using an integrative approach. We also generated new COI barcodes for Trichophoromyia auraensis, Nyssomyia shawi, and Psychodopygus paraensis, which may play a role in the transmission of Leishmania spp. in the Brazilian Amazon.