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Abstract The quantity and preservation of carbon‐rich organic matter (OM) underlying permafrost uplands, and the evolution of carbon accumulation with millennial climate change, are large sources of uncertainty in carbon cycle feedbacks on climate change. We investigated permafrost OM accumulation and degradation over the Holocene using a transect of sediment cores dating back to at least c. 6 ka, from a hillslope in the Eight Mile Lake watershed, central Alaska. We find decimeter‐scale organic‐rich (111 ± 45 kg C m⁻³) and organic‐poor (49 ± 30 kg C m⁻³) layers below an upper peat, which store 35% ± 11% and 41% ± 20% of the carbon in the upper 1 m, respectively. In organic‐poor layers, scattered¹⁴C ages of plant macrofossils and higher percentages of degradedAlnusandBetulapollen indicate reworking by cryoturbation and hillslope processes. Whereas organic carbon to nitrogen ratios generally indicate OM freshening up‐core, amino acid bacterial biomarkers, includingd‐enantiomers and gamma‐aminobutyric acid, suggest enhanced degradation prior to 5 ka. Carbon accumulation rates increased from ∼4 to 14 g C m⁻² year⁻¹from c. 8 to 0.2 ka, coinciding with decreasing temperatures and increasing moisture regionally, which may have promoted OM accumulation. Carbon stocks within the upper 1 m average 66 ± 13 kg C m⁻², varying from 77 kg C m⁻²in a buried depression on the upper slope to 48 kg C m⁻²downslope. We conclude that heterogeneity in preserved OM reflects a combination of hillslope geomorphic processes, cryoturbation, and climatic variations over the Holocene.