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Liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) were used to provide qualitative and quantitative information about microcystin (MC) congeners in western Lake Erie. Samples were collected at eight open-water locations on selected days during harmful algal blooms (HABs) in 2016 and 2017. Seven MCs were identified and 20 MCs were tentatively identified using high-resolution mass accuracies and a unique fragment (Adda m/z 135). The most abundant MC was MC-LR, followed by MC-RR, MC-YR, and MC-LA, and these congeners were quantified. Total (extracellular and intracellular) MC concentrations ranged from 0.068 to 14.88 µg/L in 2016, and from 0.050 to 10.15 µg/L in 2017, with averages of 2.71 and 1.86 µg/L, respectively. Near-shore sites in Lake Erie had higher MC concentrations and Microcystis biovolumes than off-shore sites. This implies that nutrient loading from the Maumee River greatly influences Maumee Bay, and this influence decreases with distance from the river. Consequently, six MCs (MC-LR, MC-RR, MC-LA, MC-YR, MC-LW, and MC-LF) were quantified in water samples collected from the Maumee River and the Maumee Bay shore of Lake Erie in 2017, and MC-RR was the most abundant. The total MC concentrations in river samples ranged from 0.17 to 305.03 µg/L. Additionally, an MC degradation product (linear MC-LR) was detected at all open-water locations, and data indicated an increase in its concentration towards the end of the bloom. The trends for 2016 and 2017 HABs are comparable in terms of spatial distribution and MC congeners produced, though the intensity and peak dates change.