AbstractMicrobial aerobic methane oxidation is an important sink for aquatic methane worldwide. Despite its importance to global methane fluxes, few aerobic methane oxidation rates have been obtained in freshwater or marine environments without imposing changes to the microbial community through use of ex situ methods. A novel in situ incubation method for continuous time‐series measurements was used in Jordan Lake, North Carolina, during 2020–2021, to determine reaction kinetics for aerobic methane oxidation rates across a wide range of naturally varying methane (55–1833 nM) and dissolved oxygen (DO; 28–366 μM) concentrations and temperatures (17–30°C). Methane oxidation began immediately at the start of each of 21 incubations and methane oxidation rates were 1st order with respect to methane. The data density allowed for accurate calculation of 1st‐order rate constants, k, that ranged from 0.018 to 0.462 h−1 (R2 > 0.967). Addition of ammonium (20–45 μM) to natural concentrations ranging from 0.057 to 2.4 μM did not change aerobic methane oxidation rate kinetics, suggesting that the natural population of aerobic methane oxidizers in this eutrophic lake was not nitrogen limited. Values of k inversely correlated most strongly with initial DO concentrations (R2 = 0.82) rather than temperature. Values for k increased with Julian day throughout our sampling period, suggesting seasonal influences on methane oxidation via responses to geochemical changes or shifts in microbial community abundance and composition. These experiments demonstrate a high variability in the enzymatic capacity for 1st‐order methane oxidation rates in this eutrophic lake that is tightly and inversely coupled to oxygen concentrations. Measurements of in situ aerobic methane oxidation rate constants allow for the direct quantification and modeling of the microbial community's capacity for methane oxidation over a wide range of natural methane concentrations.