Subglacial environments are largely anoxic, contain organic carbon (OC) overridden by glacier ice during periods of advance, and harbour active microbial communities. This creates favourable conditions for OC degradation via methanogenesis. It has been hypothesized that OC beneath ice sheets is converted to methane (CH4) and may be released to the atmosphere during retreat. However, there are limited data available to support this contention. Here, we present new data on the abundance, diversity and activity of methanogenic archaea and the amount and character of OC in subglacial sediments from Arctic and Antarctic glacial systems based on different substrate types. We employed long-term laboratory incubations to quantify the CH4 production potential in different subglacial settings. Significant numbers of methanogens (up to 7 × 104 cells g−1) were detected in the samples and clones of Methanomicrobiales and Methanosarcinales were identified in clone libraries. Long lag periods (up to >200 days) were observed before significant CH4 concentrations were measured. We report order of magnitude differences in rates of CH4 production (101–105 fmol g−1 d−1) in different subglacial sediments, reflecting contrasts in the origin of the sediment and the OC character. Hence, we predict that contrasting rates of CH4 production are likely to occur beneath glaciers and ice sheets that overran different types of substrate. We subsequently estimated the potential for CH4 production beneath the Laurentide/Inuitian/Cordilleran and Fennoscandian Ice Sheets during a typical 85 ka Quaternary glacial/interglacial cycle. CH4 production from lacustrine-derived OC is likely to be an order of magnitude higher (~6.3–27 Pg C) than that from overridden soils (~0.55–0.68 Pg C), possibly due to a difference in lability between lacustrine and soil OC. While representing a fraction of the entire OC pool (~418–610 Pg C), this finding highlights the importance of considering the character of different OC pools when calculating subglacial CH4 production.