Crayfish (Orconectes spp.), Asian clam (Corbicula fluminea), northern hog sucker (hog sucker; Hypentelium nigricans), and smallmouth bass (smallmouth; Micropterus dolomieu) from streams in southeastern Missouri (USA) were analyzed for total mercury (HgT) and for stable isotopes of carbon (δ¹³C), nitrogen (δ¹⁵N), and sulfur (δ³⁴S) to discern Hg transfer pathways. HgT concentrations were generally lowest in crayfish (0.005–0.112 μg/g dw) and highest in smallmouth (0.093–4.041 μg/g dw), as was δ¹⁵N. HgT was also lower and δ¹⁵N was higher in all biota from a stream draining a more heavily populated historical lead–zinc mining area than from similar sites with mostly undeveloped forested watersheds. δ¹³C in biota was lowest at spring-influenced sites, reflecting CO₂ inputs and temperature influences, and δ³⁴S increased from south to north in all taxa. However, HgT was not strongly correlated with either δ¹³C or δ³⁴S in biota. Trophic position (TP) computed from crayfish δ¹⁵N was lower in hog suckers (mean=2.8) than in smallmouth (mean=3.2), but not at all sites. HgT, δ¹³C, δ³⁴S, and TP in hog suckers increased with total length (length) at some sites, indicating site-specific ontogenetic diet shifts. Changes with length were less evident in smallmouth. Length-adjusted HgT site means in both species were strongly correlated with HgT in crayfish (r²=0.97, P<0.01), but not with HgT in Corbicula (r²=0.02, P>0.05). ANCOVA and regression models incorporating only TP and, for hog suckers, length, accurately and precisely predicted HgT concentrations in both fish species from all locations. Although low compared to many areas of the USA, HgT (and therefore methylmercury) concentrations in smallmouth and hog suckers are sufficiently high to represent a threat to human health and wildlife. Our data indicate that in Ozark streams, Hg concentrations in crayfish are at least partly determined by their diet, with concentrations in hog suckers, smallmouth, and possibly other higher-level consumers largely determined by concentrations in crayfish and other primary and secondary consumers, fish growth rates, and TP.