Monomethyl mercury (MMHg) and total mercury (THg) concentrations and Hg stable isotope ratios (δ(202)Hg and Δ(199)Hg) 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 3870ng/g) and δ(202)Hg (-1.69 to -0.20‰) reflecting the heterogeneity of Hg mining sources in sediment. The δ(202)Hg 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 (δ(202)Hg and Δ(199)Hg) 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 δ(202)Hg of pre-photodegraded MMHg and sediment IHg. Cache Creek had mass dependent fractionation (MDF; δ(202)Hg) 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.