Lutzomyia gomezi, a suspected vector of cutaneous leishmaniasis in Colombia is recorded for natural infection of Leishmania parasites, its anthrophilic behaviour and significant abundance supports its vectorial role. The difficulties associated with taxonomic identification due to lack of males require the use of molecular markers (DNA barcodes), which allows us to distinguish the species. In this study, the cytochrome oxidase I fragment was proposed as DNA barcode to identify specimens collected from Cordoba, Colombia (Planeta Rica: Arenoso/ Centro Alegre, Sahagun: Santiago Abajo/San Andresito) by using protocols for DNA extraction, PCR, and sequencing. These sequences allowed for testing the genetic diversity, genetic distance, population structure and gene flow. A phylogenetic analysis was performed with sequences registered in Genbank for this and related species such as Lutzomyia lichyi, Lutzomyia longipalpis and Lutzomyia bifoliata. In total, 24 PCR products were sequenced, resulting in an alignment of 677 nt in length, and 9 haplotypes were identified for L. gomezi. Values for polymorphic sites, haplotype and nucleotide diversity were high for specimens belonging to Sahagun and Planeta Rica. The genetic distances (TN93) and localities studied were significant (0,011-0,024), FST evidenced with mild and significant structure (0,10-0,52) and limited genetic flow (Nm=0,45-24,5). The phylogenetic analysis showns three lineages with significant distances (0,026-0,48) and sympatric between haplotypes from different zones; however, the limited sampling size and the absence of specimens belonging to other Colombian geographic areas implied more lineages. The DNA barcode methodology can answer questions about phylogeography, vector competence and genetic structuration between populations using a common marker for the scientific community.