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A variety of hierarchical gene flow models have been proposed to explain the distribution of genetic variation in aquatic environments. These models, including the Stream Hierarchy (SH), Death Valley (DV), Headwater (HW), and Widespread Gene flow (WG) models, provide testable hypotheses that focus on the degree of within-basin or within-stream network connectivity and an organism's dispersal abilities. We tested these models using Tlaloc hildebrandi (Profundulidae, Cyprinodontiformes), a freshwater fish endemic to the highlands of the Grijalva and Usumacinta River basins in southern Mexico. Data from ultraconserved elements (UCEs) showed that although T. hildebrandi was recovered as monophyletic, the sub-basins were not. We generated single nucleotide polymorphisms from the UCEs to analyse the data in a population genetic framework. These results differed between analyses such that two (STRUCTURE analysis) or three (DAPC analysis) genetic groupings were recovered. Overall, the results of this study provide support for the SH model. Some individuals from the Jataté sub-basin, however, conformed to the HW model, due to historical connections among headwater streams and rivers of the Jataté and Amarillo–Chenalhó clades/clusters. The greatest degree of gene flow has occurred from the Grijlava to the Usumacinta basins, two geographically proximate basins that have been hypothesized to have had previous connections. [ABSTRACT FROM AUTHOR]