Due to widespread atmospheric deposition of mercury (Hg), all aquatic food webs are contaminated with toxic methyl mercury (MeHg). At high concentrations, MeHg poses a health hazard to wildlife and humans. Spiders feeding in riparian habitats (hereafter referred to as riparian spiders) have been proposed as sentinels of MeHg contamination of aquatic systems. Riparian spiders are exposed to MeHg through their diets, and the concentration of MeHg in spiders is positively related to the proportion of MeHg‐contaminated emergent aquatic insects in their diets. The use of spiders as sentinels is complex because their MeHg concentrations can vary, not only among ecosystems but also between different spider taxa and as a function of spider body size. The objective of the present study was to examine how the level of ecosystem contamination, spider taxon, and spider body size interact to influence MeHg concentrations in four genera of riparian spiders from two rivers with different levels of Hg contamination. We collected four genera of riparian spiders (Tetragnatha sp., Larinioides sp., Pardosa sp., and Rabidosa sp.) from two sites along both the Clear Fork of the Trinity River and the West Fork of the Trinity River (Fort Worth, TX, USA). We analyzed concentrations of MeHg in different body sizes of spiders from each genus. We found that MeHg contamination of the river ecosystem, spider taxon, and spider body size were important determinants of MeHg concentration in riparian spiders. The results suggest that any of the four taxa of riparian spiders from the present study could be used as sentinels of aquatic MeHg contamination, but they should not be used interchangeably because of the interdependence between the effects of ecosystem contamination level, spider taxon, and body size. Future studies utilizing riparian spiders as sentinels of biomagnifying aquatic contaminants (e.g., MeHg, polychlorinated biphenyls) should consider the potentially complex interaction effects between ecosystem contamination level, spider taxon, and spider body size. Environ Toxicol Chem 2024;00:1–7. © 2024 The Author(s). Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.