Spawning aggregations of five species of catostomids were observed on the two mid-channel gravel bars of the Savannah River, Georgia and South Carolina, in 2004 and 2005 to assess the degree of spatial and temporal overlap in the use of this habitat and determine the habitat preferences leading to segregation. Spawning catostomids showed a considerable amount of temporal overlap in their use of these mid-channel gravel bars. The observed temporal overlap was consistent between 2004 and 2005 and corresponded to temperatures at which species were present. The distribution of catostomids was not uniform at the upstream gravel bar. Carpsuckers Carpiodes sp., spotted sucker Minytrema melanops and robust redhorse Moxostoma robustum both demonstrated some spatial overlap with notchlip redhorse Moxostoma collapsum; however, their overall distributions were different from one another. Northern hogsucker Hypentelium nigricans was present across the gravel bars, apparently as an egg predator. Spawning catostomids segregated based on flow, depth, slope and substratum size. Whether due to limited habitat availability or changes in the timing of reproduction due to altered cues, temporal and spatial overlap occurs between spawning catostomids despite the apparent partitioning of available spawning habitat. It is unclear, however, if this overlap results in excessive mortality in the early life-history stages of these species. Results suggest spatial overlap among catostomid species was minimized due to species spawning in areas within a narrow range of conditions. Intraspecific interactions such as nest site superimposition or disturbance may be a concern. ?? 2007 The Fisheries Society of the British Isles.