Monomethyl mercury (MMHg) and total mercury (THg) concentrations and Hg stable isotope ratios (δ²⁰²Hg and Δ¹⁹⁹Hg) were measured in sediment and aquatic organisms from Cache Creek (California Coast Range) and Yolo Bypass (Sacramento Valley). Cache Creek sediment had a large range in THg (87 to 3870 ng/g) and δ²⁰²Hg (−1.69 to −0.20‰) reflecting the heterogeneity of Hg mining sources in sediment. The δ²⁰²Hg of Yolo Bypass wetland sediment suggests a mixture of high and low THg sediment sources. Relationships between %MMHg (the percent ratio of MMHg to THg) and Hg isotope values (δ²⁰²Hg and Δ¹⁹⁹Hg) in fish and macroinvertebrates were used to identify and estimate the isotopic composition of MMHg. Deviation from linear relationships was found between %MMHg and Hg isotope values, which is indicative of the bioaccumulation of isotopically distinct pools of MMHg. The isotopic composition of pre-photodegraded MMHg (i.e., subtracting fractionation from photochemical reactions) was estimated and contrasting relationships were observed between the estimated δ²⁰²Hg of pre-photodegraded MMHg and sediment IHg. Cache Creek had mass dependent fractionation (MDF; δ²⁰²Hg) of at least −0.4‰ whereas Yolo Bypass had MDF of +0.2 to +0.5‰. This result supports the hypothesis that Hg isotope fractionation between IHg and MMHg observed in rivers (−MDF) is unique compared to +MDF observed in non-flowing water environments such as wetlands, lakes, and the coastal ocean.