Hg accumulation in marine organisms depends strongly on in situ water or sediment biogeochemistry and levels of Hg pollution. To predict the rates of Hg exposure in human communities, it is important to understand Hg assimilation and processing within commercially harvested marine fish, like the European seabass Dicentrarchus labrax. Previously, values of Δ199Hg and δ202Hg in muscle tissue successfully discriminated between seven populations of European seabass. In the present study, a multi-tissue approach was developed to assess the underlying processes behind such discrimination. We determined total Hg content (THg), the proportion of monomethyl-Hg (%MeHg), and Hg isotopic composition (e.g. Δ199Hg and δ202Hg) in seabass liver. We compared this to the previously published data on muscle tissue and local anthropogenic Hg inputs. The first important finding of this study showed an increase of both %MeHg and δ202Hg values in muscle compared to liver in all populations, suggesting the occurrence of internal MeHg demethylation in seabass. This is the first evidence of such a process occurring in this species. Values for mass-dependent (MDF, δ202Hg) and mass-independent (MIF, Δ199Hg) isotopic fractionation in liver and muscle accorded with data observed in estuarine fish (MDF, 0–1‰ and MIF, 0–0.7‰). Black Sea seabass stood out from other regions, presenting higher MIF values (≈1.5‰) in muscle and very low MDF (≈-1‰) in liver. This second finding suggests that under low Hg bioaccumulation, Hg isotopic composition may allow the detection of a shift in the habitat use of juvenile fish, such as for first-year Black Sea seabass. Our study supports the multi-tissue approach as a valid tool for refining the analysis of Hg sourcing and metabolism in a marine fish. The study's major outcome indicates that Hg levels of pollution and fish foraging location are the main factors influencing Hg species accumulation and isotopic fractionation in the organisms.