<p>Geographical features and climatic oscillations that occurred in the Quaternary are among the main factors shaping the genetic diversity of both terrestrial and aquatic taxa. Rivers are naturally discontinuous and heterogeneous habitats due to interruptions and connections of water flow that resulted from tectonic activity and Pleistocene climatic fluctuations. These factors, in combination with the biological characteristics of the species, can affect the dispersal of organisms that are restricted to rivers, which can, in turn, influence their genetic diversity, genetic structure, and gene flow. Here, we investigate the phylogeographic patterns and demographic history of the high-backed pygmy swordtail fish <i>Xiphophorus multilineatus</i> to understand the effects that geography and climatic fluctuations of the Pleistocene had on the genetic patterns of this species. We amplified the control region of the mitochondrial DNA of 62 individuals from six sites in the Tampaón basin (the only distribution area of the species) in San Luis Potosí, Mexico. We found relatively high genetic diversity and limited genetic structure, as well as demographic expansions occurring during glacial and interglacial periods of the Pleistocene. The phylogeographic patterns exhibited by <i>X. multilineatus</i> seem to reflect cycles of isolation and connection of its populations as a result of the climatic oscillations that occurred during the Pleistocene. This study constitutes a first approximation of geographic patterns of genetic variation in the high-backed pygmy swordtail fish <i>Xiphophorus multilineatus.</i></p>