Patterns of spatial variation of mercury and methylmercury (MeHg) were examined in sediments and muscle tissue of largemouth bass (Micropterus salmoides) from Amistad International Reservoir, a large and hydrologically complex subtropical water body in the Rio Grande drainage. The distributions of both Hg and MeHg were compared with environmental and biological factors known to influence production of MeHg. The highest concentrations of total Hg (THg) in sediment were found in the Rio Grande arm of the reservoir, whereas MeHg was highest at sites in the Devils River arm and inundated Pecos River (often more than 3.0 ng/g). Conditions in the sediments of the Devils River arm and Pecos River channel were likely more favorable to the production of MeHg, with higher sediment porewater dissolved organic carbon, and porewater sulfate levels in the optimal range for methylation. Although the detection of different groups of sulfate-reducing bacteria by polymerase chain reaction (PCR) was generally correlated with MeHg concentrations, bacterial counts via fluorescent in situ hybridization (FISH) did not correlate with MeHg. A sample of 156 largemouth bass (<30 cm) showed a spatial pattern similar to that of MeHg in sediments, where fish from the Devils River arm of the reservoir had higher muscle Hg concentrations than those collected in the Rio Grande arm. In 88 bass of legal sport fishing size (>35 cm), 77% exceeded the 0.3 mg/kg U.S. Environmental Protection Agency screening value. This study shows that significant variation in sediment MeHg and biotic Hg concentration can exist within lakes and reservoirs and that it can correspond to variation in environmental conditions and Hg methylation.