Abstract Cladocera serve as important bio‐ and paleo‐indicators of lake food webs and environmental conditions. The ecological optima of cladocerans are often established by regional‐scale calibration sets, with subsequent comparisons to limnological variables. However, due to logistical constraints when sampling large numbers of lakes, this approach often limits the length of the environmental gradients that can be examined. To extend spatial and limnological gradients, we combined 20 datasets (388 lakes) containing both cladoceran and environmental data, spanning multiple ecoregions across Canada and Alaska. These data were collected over c. 20 years using similar techniques in a single laboratory. Our continental‐scale analysis examined the main chemical and physical variables that influenced cladoceran assemblages, and identified critical environmental thresholds structuring assemblages. Multivariate analyses were used to examine the influences of six environmental variables (depth [either maximum depth or coring depth, which are very similar], surface area, pH, calcium [Ca], total phosphorus [TP], and dissolved organic carbon) and ecoregion classifications on cladoceran assemblages. Gradients of pH and Ca were strongly related. Daphnia longispina spp. and Chydorus brevilabris/biovatus were associated with higher pH and Ca concentrations, while Bosmina spp. and Holopedium spp. were more common in lakes with lower pH and Ca. Dissolved organic carbon was highly correlated with TP. Chydorus brevilabris/biovatus was associated with higher‐nutrient systems. Littoral chydorids, D. longispina spp., Holopedium spp., and Daphnia pulex spp. were associated with intermediate TP and dissolved organic carbon concentrations, and Bosmina spp. was more closely associated with oligotrophic systems. Physical limnological variables influenced taxonomic composition on a continental scale. For example, D. longispina spp., D. pulex spp., Bosmina spp., and Holopedium spp. were associated with larger and/or deeper systems, and littoral chydorids were associated with smaller and/or shallower lakes. A multivariate regression tree identified two thresholds important for structuring assemblages based on pH (<7 and ≥7, probably closely tied to lakewater Ca concentrations) and depth (<6.45 m and ≥6.45 m at pH < 7, and <4.75 m and ≥4.75 m at pH ≥ 7). Our results highlight the sensitivity of several cladoceran taxa to multiple chemical and physical gradients. We demonstrate that the ecological responses of cladocerans to environmental variables—often established through regional‐scale surveys—are applicable across ecoregions and broad limnological gradients, reinforcing the value of cladocerans as bioindicators of key chemical and physical variables in freshwaters and as paleoindicators for assessing human impacts on aquatic systems through time.