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Lakes are generally supersaturated in carbon dioxide (CO2) and emitters of CO2 to the atmosphere. However, estimates of CO2 flux ((Formula presented.)) from lakes are seldom based on direct flux measurements and usually do not account for nighttime emissions, yielding risk of biased assessments. Here, we present direct (Formula presented.) measurements from automated floating chambers collected every 2–3 hr and spanning 115 24 hr periods in three boreal lakes during summer stratification and before and after autumn mixing in the most eutrophic lake of these. We observed 40%–67% higher mean (Formula presented.) in daytime during periods of surface water CO2 supersaturation in all lakes. Day-night differences in wind speed were correlated with the day-night (Formula presented.) differences in the two larger lakes, but in the smallest and most wind-sheltered lake peaks of (Formula presented.) coincided with low-winds at night. During stratification in the eutrophic lake, CO2 was near equilibrium and diel variability of (Formula presented.) insignificant, but after autumn mixing (Formula presented.) was high with distinct diel variability making this lake a net CO2 source on an annual basis. We found that extrapolating daytime measurements to 24 hr periods overestimated (Formula presented.) by up to 30%, whereas extrapolating measurements from the stratified period to annual rates in the eutrophic lake underestimated (Formula presented.) by 86%. This shows the importance of accounting for diel and seasonal variability in lake CO2 emission estimates.