The present study combines data of microbial assemblages with high-resolution paleoceanographic records from Core GC1 recovered in the Chukchi Sea. For the first time, we have demonstrated that microbial habitat preferences are closely linked to Holocene paleoclimate records, and found geological, geochemical, and microbiological evidence for the inference of the sulphate-methane transition zone (SMTZ) in the Chukchi Sea. In Core GC1, the layer of maximum crenarchaeol concentration was localized surrounding the SMTZ. The vertically distributed predominant populations of Gammaproteobacteria and Marine Group II Euryarchaeota (MG-II) were consistent with patterns of the known global SMTZs. MG-II was the most prominent archaeal group, even within the layer of elevated concentrations of crenarchaeol, an archaeal lipid biomarker most commonly used for Marine Group I Thaumarchaeota (MG-I). The distribution of MG-I and MG-II in Core GC1, as opposed to the potential contribution of MG-I to the marine tetraether lipid pool, suggests that the application of glycerol dibiphytanyl glycerol tetraethers (GDGT)-based proxies needs to be carefully considered in the subsurface sediments owing to the many unknowns of crenarchaeol. In conclusion, microbiological profiles integrated with geological records seem to be useful for tracking microbial habitat preference, which reflect climate-triggered changes from the paleodepositional environment.