Mercury (Hg) contamination of aquatic ecosystems is a global environmental problem. Data are abundant on Hg contamination and factors that affect its bioaccumulation in lake communities, but comparatively little information on riverine ecosystems exists. The present study examines fish Hg concentrations of the Lower Rio Grande/Rio Bravo del Norte drainage, Texas, USA and several of its major tributaries in order to assess whether spatial variation occurs in fish Hg concentrations in the drainage and if patterns of Hg contamination of fish are related to gradients in environmental factors thought to affect Hg concentrations in fish communities. Fish, invertebrates, sediments, and water quality parameters were sampled at 12 sites along the lower Rio Grande/Rio Bravo del Norte drainage multiple times over a one-year period. Spatial variation was significant in fish Hg concentrations when fish were grouped by literature-defined trophic guilds or as stable isotope-defined trophic levels, with highest concentrations found in the Big Bend region of the drainage. Mercury in fish in most trophic guilds and trophic levels were positively related to environmental factors thought to affect Hg in fish, including water column dissolved organic carbon (DOC) and sediment Hg concentrations. It is likely that fish Hg concentrations in the Big Bend region are relatively high because this section of the river has abundant geologic Hg sources and environmental conditions which may make it sensitive to Hg inputs (i.e., high DOC, variable water levels). Results from the present study indicate that Hg contamination of the Rio Grande/Rio Bravo del Norte has substantial implications for management and protection of native small-bodied obligate riverine fish, many of which are imperiled.