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The concentration of dissolved oxygen (DO) is an important attribute of aquatic ecosystems, influencing habitat, drinking water quality, biodiversity, nutrient biogeochemistry, and greenhouse gas emissions. While average summer DO concentrations are declining in lakes across the temperate zone, much remains unknown about seasonal factors contributing to deepwater DO losses. It is unclear whether declines are related to increasing rates of seasonal DO depletion or changes in seasonal stratification that limit re‐oxygenation of deep waters. Furthermore, despite the presence of important biological and ecological DO thresholds, there has been no large‐scale assessment of changes in the amount of habitat crossing these thresholds, limiting the ability to understand the consequences of observed DO losses. We used a dataset from >400 widely distributed lakes to identify the drivers of DO losses and quantify the frequency and volume of lake water crossing biologically and ecologically important threshold concentrations ranging from 5 to 0.5 mg/L. Our results show that while there were no consistent changes over time in seasonal DO depletion rates, over three‐quarters of lakes exhibited an increase in the duration of stratification, providing more time for seasonal deepwater DO depletion to occur. As a result, most lakes have experienced summertime increases in the amount of water below all examined thresholds in deepwater DO concentration, with increases in the proportion of the water column below thresholds ranging between 0.9% and 1.7% per decade. In the 30‐day period preceding the end of stratification, increases were greater at >2.2% per decade and >70% of analyzed lakes experienced increases in the amount of oxygen‐depleted water. These results indicate ongoing climate‐induced increases in the duration of stratification have already contributed to reduction of habitat for many species, likely increased internal nutrient loading, and otherwise altered lake chemistry. Future warming is likely to exacerbate these trends.