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Abstract Rivers are prominent landscape features, acting as key promoters of diversification among freshwater organisms. Albeit generally considered potential barriers to species movement, they may also facilitate gene flow and structure populations of semiaquatic species (Riverine Thruway Hypothesis, RTH). We evaluated the role of rivers on the processes responsible for current genetic variation in the semiaquatic frog Pseudis bolbodactyla, testing whether each hydrographic basin harbours distinct genetic lineages. We sequenced three markers on 166 samples from 13 localities along the Paraná (PR), Araguaia–Tocantins (AT), and São Francisco (SF) River basins in Brazil. We recovered three populations geographically matching each hydrographic basin. Our results indicate migration among basins, with the best model selected using approximate Bayesian computation, including migration between AT and SF and ancient gene flow from PR to the AT–SF ancestor. Our findings are likely related to the orogenic events in Central Brazil dating to the Late Miocene (5 Mya), when hydrographic basins and the geomorphological features of the Brazilian Shield were formed. This suggests that P. bolbodactyla probably represents a species complex, with each lineage occurring in a distinct hydrographic basin, matching the predictions of the RTH.