During a study of microhabitat use by gilt darters (Percina evides), we compared two methods for quantifying microhabitat availability in a southern Appalachian stream (USA). The first method used stratified random sampling throughout the site and the second involved taking constrained random measurements within a 2-m radius of the focal fish. Darters were generally over-represented in microhabitats with higher average velocities, greater amounts of erosional substrata, and lower amounts of depositional and large substrata. The two methods generally yielded similar patterns of microhabitat use. Nonetheless, of the seven microhabitat categories in which differential microhabitat use occurred in summer, four were present in both data sets, but three differed between methods. We observed no differences between methods for autumn data. Finally, the standard deviations of the summer-stratified random data set were significantly greater (sign test, P < 0.05) than those of the constrained data set. Our results suggest that either method for quantifying microhabitat availability can be used to quantify the general habitat use patterns of this species, but constrained analyses yielded a more restricted view of the total habitat available. Nonetheless, if the fishes range over a site, clearly stratified habitat availability analysis is preferred.