Large rivers are usually associated with vicariance and diversification of terrestrial species, but little is known about these effects in overlooked areas from South America, like Sao Francisco River (SFR). In this study we tested this hypothesis by analyzing populations of Melipona mandacaia, an endemic stingless bee from Caatinga whose distribution is associated to SFR. The influence of the river width as a natural barrier to the gene flow among populations of M. mandacaia from both margins of SFR was inferred from genetic and morphometric data. Eleven microsatellite loci were amplified and 813 anterior right wings were used for geometric morphometrics. Most of the genetic variation (96.7%) in AMOVA was found within populations with evidence of low structuring (Fst = 0.032, p < 0.05). The Bayesian model and spatial analysis corroborated the lack of conspicuous genetic differentiation among samples. In morphometric analyses, the first four canonical variables explained 80.49% of the total variation. The cross-validation between populations from left and right margins revealed that about 55.44% of colonies were classified correctly. Thus, we conclude that the SFR should not be considered a natural geographic barrier to the gene flow in M. mandacaia.Implications for insect conservationEstimates of genetic diversity and the lack genetic structure among populations separated by a putative geographic barrier are useful to the management and conservation strategies of stingless bees, particularly in relation to the translocation of bees and colonies from one site to another.