Our goal was to build a geographic information system (GIS) tool to enhance modeling and hypothesis testing relevant to watersheds and fish fauna of the Red and Sabine Rivers in the southeastern United States. Species of concern were identified from wildlife action plans and Web sites. Spatial distributions of fish species and mercury in fillets were delineated using data from states. Public georeferenced data were obtained on land cover, soil type, forest canopy, impervious surfaces, wastewater facilities and 303(d) impaired waters. Overlay maps highlighted patterns across 8-digit hydrologic unit codes (HUCs). Bossier City, Louisiana and Beaumont, Texas areas displayed impervious surfaces over 10% and 303(d) waters per HUC were 20% and 8%, respectively. Because bowfin (Amia calva) (n=299) and bass (Micropterus spp.) (n=1493) occurred in up to 44% of HUCs and fillets contained elemental mercury concentrations across ranges monitored, they were appropriate indicators of bioavailable mercury. Of the total fish number showing >0.5ppm, 81% of records were derived from bowfin and bass, and stepwise multiple linear regressions indicated fish with mercury at these concentrations correlated with environmental variables. Detrended correspondence analysis showed total species occurrence and environmental relationships significant, where 81.6% of the variability in fish occurrence was explained by impervious surface, land cover other than canopy or impervious surface (such as wetlands and agricultural area) and canopy (forest type). Two-way indicator species analysis delineated species co-occurrence in HUCs (14 groups) and similarity of species composition (nine groups). Results identified three HUC groupings as potential targets for managerial interest. Quality control concerns for GIS development included site name data and priority rankings of critical fish species. This tool can be used to support modeling and trend analyses for several purposes, such as those relevant for developing and reporting on water quality standards and critical habitat assessments.