Stable isotopes of hydrogen (δ2H) and oxygen (δ18O) provide important quantitative measures of lake hydrology and water balance, particularly in lakes where monitoring of fluxes is incomplete. However, little is known of the relative effects of seasonal variation in water isotopes on estimates of lake hydrology, particularly over decadal scales. To address this gap, we measured water isotopes bi-weekly May-September during 2003–2016 in seven riverine lakes within the 52,000 km2 Qu’Appelle River drainage basin of the Canadian Prairies. Analyses revealed that within-year variation in δ18O values routinely exceeded that among years, reflecting rapid changes in water source, particularly in lakes with water residence times <1 year. Isotopic variation was greatest during spring following snowmelt, except in large deep lakes which exhibited limited differences among seasons or years. In contrast, large hydrological events (e.g., 1-in-140-year flood in 2011) homogenized isotopic values, even among riverine lakes separated by over 150 km, and exerted particularly strong legacy effects on large lakes. Overall, study lakes exhibited a strongly positive moisture balance (evaporation < inflow), despite regional precipitation deficits of 30 cm yr−1, with greater reliance on rainfall (vs. snow) and possibly evaporation in downstream lakes within more humid regions. We conclude that seasonal samples of water isotopes are required to characterize the hydrology of shallow lakes, or those with unknown reliance on snowmelt waters, as well as to better quantify lake susceptibility to climate variability.