Since 1978, the Lake Ontario benthic preyfish survey has provided information on the status and trends of the benthic preyfish community related to Lake Ontario Fish Community Objectives that include understanding preyfish population dynamics and community diversity. Beginning in 2015, the benthic preyfish survey expanded from US-only to incorporate lake-wide sampling sites which drastically increased the survey’s spatial coverage. In 2020, the collaborative benthic preyfish survey completed 82 bottom trawl tows across main lake and embayment sites at depths from 6 to 226 m. Compared to previous years, the survey was largely confined to ports east of Rochester on the US side, and east of Wesleyville on the Canadian side due to COVID-19 travel constraints. In total, the survey sampled 109,315 fish from 32 species in 2020. Round goby was the most numerically abundant species comprising 59% of total catch, followed by alewife (27%), and deepwater sculpin (7%). Despite being a common species represented in historical catches, slimy sculpin comprised only 0.05% of the total catch, with only 53 fish sampled in 2020. Annual biomass indices were among the lowest reported for slimy sculpin across the entire time series, whereas deepwater sculpin biomass remained high but showed a decrease from values observed in 2019. Deepwater sculpin condition in 2020 was lower than in 2019 but similar to values observed in 2017-2018. Community diversity remained high relative to historically lower values when round goby and deepwater sculpin were not captured in trawl catches. Round goby biomass in 2020 was the highest on the US time series, but not on the lake-wide series, highlighting the need to resume lake-wide sampling to further understand how regional vs lake-wide sampling affects benthic preyfish population estimates.