The southeastern United States harbors the greatest diversity of crayfish in North America, yet this biodiversity is increasingly threatened by land-use change and habitat alteration. Many species exhibit extreme endemism, often restricted to single river systems, making them especially vulnerable to urbanization, forestry, and hydrologic shifts. Despite this, few studies have examined the ecological drivers shaping crayfish assemblages in inter-ecosystem rivers. We investigated crayfish assemblage structures in the Ogeechee River Basin, Georgia, using land cover, hydrological, spatial, and phylogenetic predictors. Gradient-forest analysis identified key environmental variables, which were tested with Threshold Indicator Taxa ANalysis (TITAN) and incorporated into a Hierarchical Model of Species Communities (HMSC). Our results show that hydrologic subunits (HUC8), wetland cover, and upstream channel length are the strongest predictors of crayfish assemblage composition, contributing to a cross-validated model with a mean AUC of 0.7. Wetland cover was particularly influential, suggesting that wetlands act as critical refugia for some species and, for a select few, as a barrier to dispersal across the basin. Hydrologic subunits structured assemblages along the river continuum, while channel length reflected connectivity and habitat heterogeneity. Some species showed significant positive or negative associations with the model’s variables, suggesting that some species are specialists for those factors and can act as indicators of changes in those variables across the basin. We also found no significant biotic interactions among crayfish species within the basin, after accounting for variation due to the covariates used in the model. The model supports the existence of four major metacommunities across the basin, as illustrated by a community turnover plot generated from the first three PC axes of the model’s predicted presence/absence values. These findings demonstrate that crayfish assemblages in the Ogeechee are strongly shaped by a complex interplay of hydrologic and landscape features, underscoring the importance of conserving wetlands and maintaining river connectivity. By linking community composition to environmental drivers, this study provides a framework for predicting crayfish responses to ongoing land-use change and informs conservation strategies for endemic species in southeastern rivers.