In the Sonora Margin cold seep ecosystems (Gulf of California), sediments underlying microbial mats harbor high biogenic methane concentrations, fuelling various microbial communities such as abundant lineages of Anaerobic Methanotrophs (ANME). However biodiversity, distribution and metabolism of the microorganisms producing this methane remain poorly understood. In this study, measurements of methanogenesis using radiolabelled dimethylamine, bicarbonate and acetate showed that biogenic methane production in these sediments was mainly dominated by methylotrophic methanogenesis, while the proportion of autotrophic methanogenesis increased with depth. Congruently, methane production and methanogenic Archaea were detected in culture enrichments amended with trimethylamine and bicarbonate. Analyses of DGGE fingerprinting and reverse-transcribed PCR amplified 16S rRNA sequences retrieved from these enrichments revealed the presence of active methylotrophic Methanococcoides burtonii relatives and several new autotrophic Methanogenium lineages confirming the co-occurrence of Methanosarcinales and Methanomicrobiales methanogens with abundant ANME populations in the sediments of the Sonora Margin cold seeps.