Stable-isotope probing (SIP) was used to identify glucose degradation bacteria and archaea under anaerobic thermophilic condition in a municipal solid waste decomposition ecosystem. A solid waste degradation ecosystem was split in parallel digestors that were fed with 13C-labeled glucose as the main carbon source. After DNA extraction and ultracentrifugation, 16S rRNA gene sequence clones retrieved from 13C-DNA were mostly related to the genus Acetivibrio/Clostridium, genus Garciella and clustered to a group of unclassified Clostridiales (MSW Cluster II). This indicates these populations could direct assimilate glucose. The populations related to unclassified Bacteroidetes, genus Anaerobaculum, genus Thermacetogenium, unclassified Firmicutes and some unclassified Bacteria were likely involved in secondary phase of glucose degradation. The archaeal sequences retrieved from 13C-DNA belonged exclusively to the genus Methanoculleus. This hydrogenotrophic methanogen was dominant archaea in anaerobic thermophilic glucose degradation.