Bichromomyia flaviscutellata (Mangabeira, 1942) sensu stricto (Diptera: Psychodidae) has been recognized as the main vector of Leishmania amazonensis in the Brazilian Amazon. For this reason, it is of paramount importance to understand the distribution of genetic diversity of populations of this vector, particularly the genetic structure and gene flow, for its management and control efforts. This study investigated the phylogeographic structure of five B. flaviscutellata s.s. populations from the central Brazilian Amazon region by analyzing 1,141 bp fragment of the 3′ region of the COI gene. A total of 85 specimens of B. flaviscutellata s.s. were sequenced from Manaus (14), Rio Preto da Eva (10), Pitinga (14), Novo Airão (21), and Autazes (26); all in the state of Amazonas. The dataset yielded 59 haplotypes, most of them connected to each other in the main network. There were high levels of intrapopulation genetic variability (h = 0.945 ± 0.035 – 0.978 ± 0.054). The genetic distance values among populations varied from moderate (0.0873) to very high (0.3535), and all comparisons were significant, as well as the hierarchical analysis (Φ_ST = 0.2145). In contrast, these comparisons revealed a high number of shared sites (S_s = 6–34) and no difference in fixed sites (S_f = 0) among populations indicating absence of historical isolation. The Mantel test indicated that 67.92% (r = 0.6792; P = 0.06) of the genetic structure observed in B. flaviscutellata s.s. cannot be explained by the isolation-by-distance (IBD) model. This genetic structure, weakly explained by the IBD, may be due mainly by the forest habitat fragmentation and the low dispersal (flight) capacity of sand flies. Both factors could lead to population fragmentation and isolation, which promote genetic differentiation. Taken together, these findings suggest that the genetic structure observed in the studied populations of B. flaviscutellata s.s. is likely generated by microevolutionary processes acting at the population level at the present time and, therefore, evolutionary lineages were not recognized among the populations analyzed.