Photosynthetic oxidation ponds are a low-cost method for secondary treatment of wastewater using natural and more energy-efficient aeration strategies. Methane (CH4) is produced during the anaerobic digestion of organic matter, but only some of it is oxidized in the water column, with the remaining CH4 escaping into the atmosphere. In order to characterize the CH4 flux in two photosynthetic oxidation ponds in a wastewater treatment plant in northern California, the isotopic compositions and concentrations of CH4 were measured in the water column, in bubbles and in flux chambers, over a period of 12 to 21 months to account for seasonal trends in CH4 emissions. Methane flux varied seasonally throughout the year, with an annual average flux of 5.5 g CH4 m−2 d−1 Over half of the CH4 flux, 56.1–74.4% v/v, was attributed to ebullition. The oxidation efficiency of this system was estimated at 69.1%, based on stable carbon isotopes and a calculated fractionation factor of 1.028. This is the first time, to our knowledge, that a fractionation factor for CH4 oxidation has been empirically determined for oxidation ponds. Quantifying CH4 emissions from these systems is essential to properly identify their contribution and to mitigate their impact on global warming.