Background Understanding the processes that influence distribution of organisms is a major goal in evolutionary biology. Speciation in freshwater fishes is mainly associated with the “island-like” model of evolution, in which the formation of land barriers between different hydrographic basins interrupts gene flow and promotes isolation. Freshwater fish therefore provide an excellent model system for macro- and micro-evolutionary studies. The Mesa Silverside, Chirostoma jordani, is one of the most widespread freshwater fish species in the Mexican Plateau, a geologically complex physiographic region with a long history of genesis, destruction and compartmentalization of hydrographic basins that has promoted the dispersal and isolation of freshwater fishes. Methods We used mitochondrial (Cytb and D-loop) and nuclear (first intron of the ribosomal protein S7) data and used phylogeographic and coalescent based methods to elucidate the evolutionary history of C. jordani throughout its distributional range on the Mexican Plateau. Results The results obtained in the present study revealed that C. jordani consists of two main genetic groups with geographical correspondence. Clade I occur exclusively in north-western basin and shows population structure. Clade II is widely distributed across the west, central and eastern basins without population structure. The split between these two main clades was estimated at 1.4 Mya. This cladogenetic event may be associated with the allopatric process promoted by the fragmentation and compartmentalization of hydrographic basins induced by the geological and climatic history of the Mexican Plateau.