Harmful cyanobacteria blooms, and associated cyanotoxins, are a growing threat in estuarine waters as upstream blooms are exported into coastal environments. It is known that the cyanotoxin microcystin (MC) can accumulate within biota, but to date there has been little research on MC bioaccumulation in estuarine shellfish. The San Francisco Estuary aquatic food web has undergone a profound change in response to widespread colonization of aquatic invasive species such as Asian clams (Corbicula fluminea) in the freshwater portion of the estuary known as the Sacramento-San Joaquin Delta (Delta). We studied MCs accumulation in Delta Asian clams and invasive signal crayfish (Pacifastacus leniusculus), food sources to native fish species and humans, over a two-year period. We found MCs accumulate in Asian clams across all months and at all study sites, with seasonal maxima occurring during the summer. Crayfish at times also accumulated high concentrations of MCs. Although MC concentrations rarely exceeded public health advisory levels, the persistence of MCs year-round in Delta shellfish still poses a chronic risk to consumers. We expanded our study to also measure MCs further along the freshwater to marine continuum, by collecting crabs (Rhithropanopeus harrisii and Metacarcinus magister) and shrimp (Crangon franciscorum) in more saline waters, downstream of the Delta. Summertime concentrations of MC in crabs exceeded public health advisory levels at most sites and preliminary data suggests that shrimp also accumulate MCs. ELISA and LC-MS analytical methods were used to measure free and protein-bound MCs shellfish tissues. Our initial comparison of ELISA and LC-MS was highly variable due to substantial matrix effects in the LC-MS chromatograms. We found an improved MC extraction method for use when analyzing certain shellfish taxa by LC-MS showed good agreement with ELISA results. Our results highlight the widespread MC contamination of shellfish across a large estuary and that both ELISA and LC-MS are useful for detecting MCs in shellfish.