We examined microhabitat selection by the Gulf sturgeonAcipenser oxyrinchus desotoia subspecies of Atlantic sturgeon that is listed as threatened by the U.S. government. Individuals and groups of sturgeons were placed in a Ferguson flume at low (4–6 cm/s) and high (5–17 cm/s) water flows, and their distributions relative to substrate and water velocity were recorded with a video camera. A Jacobs' selectivity index (D) was used to evaluate substrate and velocity preference by the fish. Distribution of individual fish, relative to water velocity, differed significantly in the low (X2= 11.8, df = 2,P< 0.05) and high (X2= 39.0, df = 12,P< 0.05) water flows. Groups also selected velocities within low (X2= 36.1, df = 2,P< 0.05) and high (X2= 63.8, df = 12,P< 0.05) flows. Individual fish did not select substrates in low flow but moderately preferred cobble (D= 0.32) in high flow. Groups moderately preferred sand (D= 0.40) and moderately avoided cobble (D= –0.47) in low flow. No selection was observed by groups in high flow. Differences in use of substrate types by individual fish were not significant in low flow (X2= 4.43, df = 2,P≥ 0.05); however, the differences were significant in the other treatment combinations (group, low flow:X2= 134.2, df = 2,P< 0.05; group, high flow:X2= 21.05, df = 2,P< 0.05; individual, high flow:X2= 11.03, df = 2,P< 0.05). Results from this laboratory experiment have implications for future microhabitat studies and represent the first quantified look at microhabitat selection by age-0 Gulf sturgeons.