The genus Hemigrammus is one of the most representative of the Characidae family. It includes about 60 valid species, distributed mainly in the Amazon basin, but species also occur in the São Francisco River basin and in coastal drainages of the Northeast region of Brazil. This study focused on two species within this genus, Hemigrammus brevis and H. marginatus, which coexist in the Itapicuru (Northeastern Mata Atlântica ecoregion, NMAF), São Francisco (São Francisco ecoregion, SF), and potentially the Vaza-Barris (NMAF) River basins. The sharing of these species among these basins partly supports the hypothesis that the Itapicuru and Vaza-Barris rivers may have once been tributaries of a basin that included the main course of São Francisco River. Although the sharing of fish species among these ecoregions provides a starting point for understanding the biogeographic history of the rivers draining them, no comprehensive population-level study has been conducted until now. In this context, the present study aimed to investigate, describe, and analyze the phylogeographic pattern of H. brevis and H. marginatus within the NMAF and SF ecoregions. Mitochondrial COI gene sequences were obtained and analyzed from samples collected across all basins where they occur, including their type localities. The results revealed haplotype sharing among some populations of H. marginatus from different localities and ecoregions, along with subtle structuring within the NMAF. In contrast, H. brevis exhibited no haplotype sharing, indicating subtle structuring between the Itapicuru and São Francisco River basins, with only a few mutational steps separating the distinct haplotypes. These findings suggest that the two congeneric species share a similar phylogeographic history, supporting the idea of past connectivity between the SF and NMAF ecoregions, particularly through tributaries of the right bank of the São Francisco River and the Itapicuru river basin.