Thermal hydrolysis pretreatment coupled with Thermophilic Anaerobic Digestion (TAD) for Waste Activated Sludge (WAS) treatment is a promising combination to improve biodegradation kinetics during stabilization. However, to date there is a limited knowledge of the anaerobic biomass composition and its impact on TAD process performances. In this study, the structure and dynamics of the microbial communities selected in two semi-continuous anaerobic digesters, fed with untreated and thermal pretreated sludge, were investigated. The systems were operated for 250 days at different organic loading rate. 16S rRNA gene clonal analysis and Fluorescence In Situ Hybridization (FISH) analyses allowed us to identify the majority of bacterial and archaeal populations. Proteolytic Coprothermobacter spp. and hydrogenotrophic Methanothermobacter spp. living in strict syntrophic association were found to dominate in TAD process. The establishment of a syntrophic proteolytic pathway was favoured by the high temperature of the process and enhanced by the thermal pretreatment of the feeding sludge. Proteolytic activity, alone or with thermal pretreatment, occurred during TAD as proven by increasing concentration of soluble ammonia and soluble COD (sCOD) during the process. However, the availability of a readily biodegradable substrate due to pretreatment allowed to significant sCOD removals (more than 55%) corresponding to higher biogas production in the reactor fed with thermal pretreated sludge. Microbial population dynamics analysed by FISH showed that Coprothermobacter and Methanothermobacter immediately established a stable syntrophic association in the reactor fed with pretreated sludge in line with the overall improved TAD performances observed under these conditions.