Interplay between the complex geography, hydrogeomorphological history, past climatic changes, and anthropogenic pressures is likely responsible for the current diversity and species' distribution of freshwater fishes in the Iberian Peninsula. To further disentangle the evolutionary processes promoting the diversification of endemic Iberian Cyprinids through time and space, we explored the patterns of genetic diversity of the Iberian arched-mouth nase, Iberochondrostoma lemmingii (Steindachner, 1866), using molecular markers rendering at different timescales: the mitochondrial gene cytochrome b and seven microsatellite loci. Both markers showed significant differentiation of populations though the relative genetic distances among populations were different between markers. Mitochondrial DNA results indicate the isolation of hydrographic basins as the main driver of population differentiation, with Tejo as the centre of diversification. The results also support connections between Tejo, Guadiana, and Guadalquivir, with levels of divergence suggesting an earlier severance of Guadalquivir, whereas Guadiana and Tejo maintained connections until a more recent past. Establishment of more peripherial populations in small southern basins (Quarteira and Almargem) could have been ruled by founder events. However, the analysis of present-day genetic configuration suggested by microsatellite data implies, for the first time, the involvement of other factors in the evolution of arched-mouth Iberian nase populations. Relative low genetic distances between inter-basin populations (Tejo and Guadiana) and the lack of concordance between differentiation and geography suggest a possible influence of human-mediated translocations in the population genetic patterns of I. lemmingii. High intra-basin differentiation levels were found within Tejo and Guadiana and may be associated with factors intrinsic to the species (e.g. low dispersal capability) or natural and/or artificial barriers to gene flow. The low vagility of the species appears to be an important factor influencing the evolutionary processes shaping the phylogeographical patterns of I. lemmingii, which could be relevant for the conservation of this threatened species. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105, 559–572.