Fish species exhibit different preferences and are segregated according to a set of physical habitat conditions. We documented microhabitat preferences on the three dimensions (substrate type, water depth, current velocity) for two native species: the stream catfish, Trichomycterus corduvense and the eel catfish, Heptapterus mustelinus. The study was conducted on the Anizacate River in a semiarid region of Córdoba Province located in central Argentina. We established one transect perpendicular to flow at the downstream end of each study site with subsequent parallel transects spaced at 5 m intervals throughout the length of the study site. Fish collections and habitat measurements were made in 1 m2-quadrates at 3 m-intervals along each transect. We quantified available microhabitats and estimated the proportion used by both fish species through suitability curves, niche breadth and Shoener ́s overlap formula. Current velocity is the key factor that distinguishes microhabitat use between T. corduvense and H. mustelinus. The former species is acting as a velocity specialist whereas the latter could be considered as a generalist. In spite of the trophic competition between these species, spatial partitioning may be promoting coexistence of H. mustelinus and T. corduvense in Anizacate River.