Anaerobic oxidation of methane (AOM) is a considerable sink for the greenhouse gas methane (CH4) in marine systems, but the importance of this process in terrestrial systems is less clear. Lowland boreal soils and wet tropical soils are two hot spots for CH4 cycling, yet AOM has been essentially uncharacterized in these systems. We investigated AOM in soils from sites in Alaska and Puerto Rico. Isotope tracers were utilized in vitro to enable the simultaneous quantification of CH4 production and consumption without use of biological inhibitors. Boreal peat soil and tropical mineral soil oxidized small but significant quantities of CH4 to CO2 under anoxic conditions (p < 0.001). Potential AOM rates were 21 ± 2 nmol gdw−1 d−1 and 2.9 ± 0.5 nmol gdw−1 d−1 for the boreal and tropical soils, respectively. The addition of terminal electron acceptors (NO3−, Fe(III), and SO42−) inhibited AOM and methanogenesis in both soils. In all incubations, CH4 production occurred simultaneously with AOM, and CH4 production rates were always greater than AOM rates. There was a strong correlation between the quantity of CH4 produced and the amount of CH4 oxidized under anoxic conditions (Alaska: r = 0.875, p < 0.0001; Puerto Rico: r = 0.817, p < 0.0001). CH4 oxidation under anoxic conditions was biological and likely mediated by methanogenic archaea. While only a small percentage of the total CH4 produced in these soils was oxidized under anoxic conditions (0.3% and 0.8% for Alaskan and Puerto Rican Soils), this process is important to understand since it could play a measurable role in controlling net CH4 flux.