Climatic variations during the Quaternary period had a considerable impact on landscapes and habitat fragmentation (rivers) in North Africa. These historical events can have significant consequences on the genetic structure of the populations. Indeed, geographically separated and genetically isolated populations tend to differentiate themselves through time, eventually becoming distinct lineages, allowing new species to emerge in later generations. The aim of the present study is to use genetic and morphological techniques to evaluate the major role of the Saalian glaciation (Middle Quaternary) in the establishment of the geographic space and in the evolution of the intraspecific genetic diversity, by tracing the demographic history of barbels belonging to the Luciobarbus pallaryi (Cyprinidae) species in the Guir Basin (Algeria). In this context, two populations, from two distinct and isolated sites, were studied. Analysis of the cytochrome b (cyt b) mitochondrial markers and of the D-loop control region has shown that the upstream and downstream Guir populations are genetically differentiated. The molecular analyses suggest that the upstream population was disconnected from this hydrographic system during the Saalian glaciation period of the Quaternary. Subsequently, it was isolated in the foggaras underground waters in the Great Western Erg, at approximately 320 000 years BP, creating, through a bottleneck effect, a new allopatric lineage referred to as Adrar . Conversely, the high genetic diversity in the upstream Guir (Bechar) population suggests that the stock is globally in expansion. These barbels (n = 52) were also examined with meristic, morphometric, osteological, and biological features. These data also reveal a complete discrimination between the two populations, with a remarkable and distinctive behavioural adaptation for the Adrar specimens: neoteny.