In this study, we evaluated the distribution and concentrations of a range of neonicotinoid insecticides (NNIs) and other insecticides, fungicides, biocides and selected herbicides in watersheds that drain into the lower Great Lakes in Ontario, Canada. Polar Organic Chemical Integrative Samplers (POCIS) were deployed in 18 watersheds during late May to late June of 2016. Grab samples were also collected in 7 of these watersheds. There was generally good agreement between the time-weighted average concentrations of pesticides estimated from the POCIS and the concentrations detected in grab samples. The NNIs, thiamethoxam, clothianidin and imidacloprid, were present in several watersheds at concentrations that exceeded the Canadian Water Quality Guideline for imidacloprid of 0.23 μg/L. The new generation insecticides, flonicamid and flupyradifurone were also detected in some watersheds, which is the first report of these pesticides in the peer-reviewed literature. Atrazine, 2,4-D, dicamba, carbendazim, thiophanate methyl and several azole-based fungicides were also widely detected. Discriminant Function Analysis (DFA) indicated that a high proportion (i.e. >80%) of the watersheds could be discriminated from each other on the basis of the pattern of pesticides detected in surface waters, and the proportion of field crops in the watershed.