The anaerobic oxidation of non-methane hydrocarbons mediated by sulfate-reducing bacteria (SRB) is a major process of organic matter degradation at marine hydrocarbon seeps. Several SRB have been successfully cultured, however, knowledge about in situ active organisms is still very limited. Here, we identified alkane-degrading key players from two contrasting seeps at the Mediterranean Amon Mud Volcano (Amon MV) and Guaymas Basin in the Gulf of California using complementary stable-isotope probing (SIP) techniques. Anoxic sediments were incubated with 13C-labeled butane or dodecane under close to in situ conditions. DNA- and RNA-based SIP in combination with 454-pyrosequencing (PYRO-SIP) allowed the identification of four phylogenetically distinct deltaproteobacterial groups of alkane-oxidizing SRB within the family Desulfobacteraceae. We named the groups degrading short-chain alkanes ‘SCA-SRB1’ and ‘SCA-SRB2’ and those degrading long-chain alkanes ‘LCA-SRB1’ and ‘LCA-SRB2’. CARD-FISH with newly developed specific probes revealed a high relative in situ abundance of SCA-SRB1 and SCA-SRB2 with 2% of the total community, while groups LCA-SRB1 and LCA-SRB2 were below 1% of total cells. Protein-based SIP (Protein-SIP), which enables to trace stable isotopes from substrate to protein, confirmed alkane-degrading key players of the family Desulfobacteraceae. In addition, Protein-SIP indicated additional carbon sources for 13C biosynthesis besides alkanes, and gave insights into possible metabolic pathways: (1-methylalkyl)succinylation as initial step of butane degradation and the oxidative Wood–Ljungdahl pathway as terminal point of alkane degradation.