Efforts to determine the utilization of Gulf of Mexico (GOM) chemosynthetic production by benthic predators have relied on stable isotope differences between photosynthetic and chemosynthetic production. Whereas the photosynthetic δ¹³C value in GOM surface waters is relatively uniform, chemosynthetic production may differ in different areas depending on prevalence of thiotrophy versus methanotrophy and inorganic carbon source. In this paper we compare the δ¹³C and δ¹⁵N signatures of the symbiont-containing mussel, Bathymodiolus childressi Gustafson, 1998, from four different chemosynthetic sites to test the hypothesis that methanotrophic production results in significant differences among them. Bathymodiolus childressi from two areas characterized by brine seepage and biogenic methane (GC425 and GC233) had very low δ¹⁵N (−3.7‰ and −16.6‰) and δ¹³C (−57.5‰ and −63.5‰) relative to areas with substantial thiotrophic production (GC234 and GC185). Bathymodiolus childressi from each chemosynthetic community had significantly different δ¹³C, and three of the sites also had distinct δ¹⁵N values. The δ¹³C and δ¹⁵N signatures of hagfish (Eptatretus sp.) and giant isopods (Bathynomus giganteus) captured from two sites showed little or no chemosyntheic usage. Squat lobsters (Munidopsis sp.) showed heavy incorporation of chemosyntheic production, but did not directly consume B. childressi.