The aim of this study was to investigate whether methanogenic pathways and community structures are heterogeneously distributed in the Kuan-Tzu-Ling high temperature seepage using geochemical and molecular approaches. Sediments collected from two sites, the mud pool and the borehole, in the Kuan-Tzu-Ling region were incubated with and without exogenous acetate at different temperatures under anoxic conditions. Concentrations of CH4, CO2 and acetate, and δ13C of methane and CO2 were monitored through time. Analyses of mcrA gene that catalyzes the final step of methanogenesis and 16S rRNA gene sequences were also carried out to explore the assemblages of methanogenic populations. Our results showed that methane yields increased along with the acetate consumption at different rates at different temperatures regardless of sediments inoculated. Analyses of carbon isotopic compositions of methane yielded different patterns for different samples. For the samples collected from a mud pool, the δ13C values of methane increased through time at 40 to 60oC. In contrast, the δ13C values decreased through time at high temperatures. Since acetoclastic methanogenesis generally fractionates carbon isotopes at a magnitude smaller than hydrogenotrophic and methylotrophic methanogenesis does, evidence based on the isotopic compositions suggests a shift from acetoclastic to hydrogenotrophic methanogensis with the increasing temperatures. For the sample collected from a borehole that potentially penetrates to a deeper region, the δ13C values of methane decreased through time at all investigated temperatures. The variation in isotopic compositions suggests that the provided acetate was decomposed into hydrogen and carbon dioxide prior to being converted into methane. Analyses of mcrA gene yielded the sequences related to those of hydrogenotrophic methanaogens. The detected 16S rDNA sequences were related to acetoclastic and hydrogenotrophic methanogens. These observations suggestthat the two methanogenic pathways may coexist in the deeper region of the Kuan-Tzu-Ling hydrocarbon seepage, and sediments and fluids collected from two sites might entrain different microbial communities at different depths.