Ponds contribute to global and regional greenhouse gas (GHG) budgets due to ideal conditions for carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) production in their shallow waters, combined with their extensive cumulative surface area. One region with numerous natural ponds is the Canadian Prairie Pothole Region. In a large survey of 145 pothole ponds, we quantified GHG saturation and diffuse flux and explored the role of water chemistry in GHG dynamics, for the first time in the region. We found that ponds were consistently supersaturated with CH4, often supersaturated with CO2 (57% of observations), and often undersaturated with N2O (65% of observations). Methane and CO2 saturation were higher in summer than spring (p < 0.001 and p = 0.023, respectively), but N2O saturation was higher in spring (p = 0.0037). Despite large variation in physicochemical conditions across ponds in the survey, sulphate concentration and pH were good predictors of CH4 and CO2 saturation, respectively. No strong predictors were identified for N2O saturation. Notably, our analysis highlights that dissolved CH4 in low sulphate ponds (<178 mg L–1) exhibited stronger response to seasonal increases in temperature than ponds with higher sulphate. This research fills an important knowledge gap about the GHG dynamics of prairie pothole ponds and can also be used in ongoing efforts to describe ecosystem services (or disservices) of ponds in this agriculture-dominated region.