Leishmaniasis is considered one of the six most important infectious diseases in the world. In spite of its importance, the leishmaniasis is one of the world's most neglected tropical diseases. Bichromomyia flaviscutellata sensu lato is a complex composed of at least three species: B. flaviscutellata sensu stricto, B. reducta and B. olmeca. The latter is composed of three subspecies: B. olmeca olmeca, B. olmeca bicolor and B. olmeca nociva, which are distributed from Central America to South America. Of these, B. flaviscutellata s.s. is recognized as the main vector of Leishmania amazonensis in Brazil. The present study aimed to identify molecularly the species and subspecies of the B. flaviscutellata complex using the 5' region of the COI gene (Barcode region). A total of 44 specimens, comprising 22 B. flaviscutellata s.s. and 22 B. olmeca nociva, were analyzed from six localities in the Brazilian Amazon: five in the State of Amazonas (Autazes, Manaus, Pitinga, Novo Airão, and Rio Preto da Eva), and one in the State of Pará (Serra do Cachorro). Three sequences from B. olmeca olmeca and one of B. olmeca bicolor from GenBank were also added to the dataset, totaling 48 sequences with a length of 549 base pairs (bp). The total dataset generated 28 haplotypes and four disconnected networks. Phylogenetic analyses using three algorithms (Neighbor-Joining [NJ], Maximum Likelihood [ML] and Bayesian Inference [BI]) generated similar topologies and most clades were from moderately to highly supported. The phylogenetic relationship, together with genetic distance values (1%) and haplotypes networks, confirm the position of B. olmeca bicolor as a subspecies of B. olmeca olmeca. However, B. olmeca nociva was closer phylogenetically to B. flaviscutellata s.s. than to B. olmeca olmeca and B. olmeca bicolor. Additionally, the haplotype network separated B. olmeca nociva from B. olmeca olmeca and B. olmeca bicolor. These findings, combined with previous morphological data, suggest that the B. olmeca nociva should be elevated to full-species status. The findings of this study also found that B. flaviscutellata s.s. populations may be in process of forming lineages.