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In this study, a quantitative PCR (qPCR) method was applied to amplify ancient DNA (aDNA) of different methane-oxidizing bacteria (MOB) types in lake sediments and to reconstruct microbial community dynamics over the last 1200 years. We also used reconstructions of in-lake nutrients concentrations, air temperature fluctuations, and sedimentary organic matter dynamics to study impacts of past environmental and climatic changes on MOB community composition. DNA preservation in lake sediments is sufficient, and qPCR amplification was successfully applied to the analysis of MOB aDNA. Temporal changes in MOB community showed different patterns between lakes, and drivers of past MOB dynamics slightly differed between lakes and among MOB groups. Overall, MOB developments were generally correlated to proxies of organic matter quality/quantity and climate data. Moreover, our results could emphasize the importance of nutrients availability in structuring MOB community, and the higher ability of MOB type 2 to access nutrients under nitrogen/nutrients limited conditions. Therefore, our study provides an operational and time-effective method to reconstruct past CH4 oxidation in lakes and could help to identify the driving factors of past temporal dynamics of MOB community.