We examined how dietary factors recorded by C and N influence Hg uptake in 347 individuals of yellowfin tuna (Thunnus albacares), an important subsistence resource from the Galápagos Marine Reserve (GMR) and the Ecuadorian mainland coast (EMC) in 2015-2016. We found no differences in total Hg (THg) measured in red muscle between the two regions and no seasonal differences, likely due to the age of the fish and slow elimination rates of Hg. Our THg concentrations are comparable to other studies in the Pacific (0.06–2.88 mg/kg wet weight), but a subset of individuals exhibited the highest mercury concentrations yet reported in yellowfin tuna. Mercury isotope values differed between Δ199Hg and δ202Hg in both regions (Δ199Hg = 2.86±0.04‰ vs. Δ199Hg = 2.33 ± 0.07‰), likely related to shifting food webs and differing photochemical processing of Hg prior to entry into the food web. There were significantly lower values of both δ15N and δ13C in tuna from GMR (δ15N: 8.5–14.2‰, δ13C: -18.5–-16.1‰) compared to those from the EMC (δ15N: 8.3–14.4‰, δ13C: -19.4–-11.9‰), of which δ13C values suggest spatially-constrained movements of tunas. Results from the pooled analysis, without considering region, indicated that variations in δ13C and δ15N values tracked changes of Hg stable isotopes. Our data indicate that the individual tuna from this study were resident fish of each region and heavily influenced by upwellings related to the Eastern Pacific Oxygen Minimum Zone and the Humboldt Current System. C, N, and Hg isotopes reflect foraging behavior mainly on epipelagic prey in shallow waters and that food web shifts drive Hg variations between these populations of tuna.