Aquatic palynomorphs have the potential to provide valuable insights into past environments but little has been done to assess the utility of freshwater dinoflagellate cysts as palaeoecological proxies of lacustrine environments. This study is the first to compare the distribution of freshwater dinoflagellate cysts found in surface sediments from 32 boreal lakes with varying physical and chemical characteristics. Cysts of Fusiperidinium wisconsinense, Parvodinium umbonatum, and Peridinium willei were nearly ubiquitous, but of the remaining cyst taxa, only Parvodinium inconspicuum, Peridinium limbatum, and Peridinium volzii were abundant but present in a smaller number of lakes. Assemblage composition broadly clustered into three groups: 1) an assemblage (primarily Peridinium spp.) associated with relatively shallow, mesotrophic lakes; 2) an assemblage dominated by F. wisconsinense in mesotrophic lakes with intermediate depth and relatively high pH and alkalinity; and 3) an assemblage dominated by P. umbonatum in deeper oligotrophic lakes. Moreover, changes in dinoflagellate cyst assemblage were assessed in a sediment core spanning the Holocene from nearby Gall Lake and interpreted alongside other previously published paleoecological proxies from this core. High abundances of many dinoflagellate cysts including P. limbatum, P. willei, F. wisconsinense, and P. inconspicuum were observed when lake production was high at a time when lake-levels were low and climate was more arid than today. This study suggests that dinoflagellate cysts in lacustrine sediments are, when abundant, promising both in modern and core samples, and the relationships between assemblages today and in the past can contribution additional information in understanding past ecological conditions.