Cyprinids in small streams tend to segregate spatially to reduce competitive interaction and avoid predation. Understanding environmental parameters by which these fishes segregate provides valuable information on structure of fish guilds. Previous multivariate analyses of field data suggest spatial segregation occurs vertically within the water column and in relation to aquatic plants. In an experimental flume we controlled water flow and these two parameters to further investigate microhabitat partitioning of three cyprinid species: blacktail (Cyprinella venusta), bluntface (C. camura), and striped shiners (Luxilus chrysocephalus). Fish distribution relative to the environmental parameters was quantified with videography and expressed as percent occurrence. We observed significant differences across fish treatments in microhabitat selection relative to water flow and water column, and differences in species overlaps. These data support the idea that vertical position in water column is a major factor delineating resource gradients and maintaining habitat guilds. Blacktail shiners, however, showed a higher affinity to the upper water column than previously reported, and no differences were noted among the three shiners in their distributions relative to aquatic plants.