Quantifying patterns of habitat use by riverine species is logistically challenging, yet instream habitat characteristics are likely important in explaining the distribution of species. We integrated radiotelemetry and sonar habitat mapping to quantify instream habitat use by female Barbour's Map Turtles (Graptemys barbouri) in Ichawaynochaway Creek, a tributary to the Flint River. We used logistic regression and a Bayesian information-theoretic approach to evaluate habitat use relative to habitat availability based on random locations. Over the two-year study period, turtles used an average of 839±199 m of creek length and exhibited site fidelity (mean 50% kernel density  =  0.23±0.05 ha). Substrate was generally more predictive of habitat use of female G. barbouri compared to large woody debris and water depth. Turtles generally used deeper habitats close to rocky-boulder and rocky-fine substrate with greater amounts of large woody debris. Estimates of home range size and habitat use found in this study improve our understanding of the spatial ecology of G. barbouri and provide a baseline for their habitat use in a relatively undisturbed section of stream. It is imperative to understand the spatial ecology of species, such as map turtles, that are particularly vulnerable to indirect effects of habitat modifications caused by impoundments, sedimentation, pollution, and snagging.