Controls on the fate of ~277 Pg of soil organic carbon (C) stored in permafrost peatland soils remain poorly understood despite the potential for a significant positive feedback to climate change. Our objective was to quantify the temperature, moisture, organic matter, and microbial controls on soil organic carbon (SOC) losses following permafrost thaw in peat soils across Alaska. We compared the carbon dioxide (CO2) and methane (CH4) emissions from peat samples collected at active layer and permafrost depths when incubated aerobically and anaerobically at −5, −0.5, +4, and +20 °C. Temperature had a strong, positive effect on C emissions; global warming potential (GWP) was >3× larger at 20 °C than at 4 °C. Anaerobic conditions significantly reduced CO2 emissions and GWP by 47% at 20 °C but did not have a significant effect at −0.5 °C. Net anaerobic CH4 production over 30 days was 7.1 ± 2.8 μg CH4-C gC−1 at 20 °C. Cumulative CO2 emissions were related to organic matter chemistry and best predicted by the relative abundance of polysaccharides and proteins (R2 = 0.81) in SOC. Carbon emissions (CO2-C + CH4-C) from the active layer depth peat ranged from 77% larger to not significantly different than permafrost depths and varied depending on the peat type and peat decomposition stage rather than thermal state. Potential SOC losses with warming depend not only on the magnitude of temperature increase and hydrology but also organic matter quality, permafrost history, and vegetation dynamics, which will ultimately determine net radiative forcing due to permafrost thaw.