The compartmentalized anaerobic reactor (CAR) is a patent novel high-rate reactor, which consists of three compartments. The reactor has a great potential for application due to its many advantages. In this work, the microbial consortium, spatial distribution, and their relationship with performance of CAR were investigated by means of polymerase chain reaction, denaturing gradient gel electrophoresis, and fluorescence in situ hybridization. The results showed that the predominant archaea were Methanobacterium, Methanosaeta, and Methanospirillum, and the predominant bacteria were Firmicutes, Deltaproteobacteria, Spirochaetes, Actinobacteria, and Gammaproteobacteria in the microbial consortium. The methanogenic archaea (MA), the hydrogen-producing acetogenic bacteria (HAB), and the hydrolytic fermentative bacteria (HFB) were found to be predominant in the upper, middle, and bottom compartments, respectively. The results revealed that the granular sludge took on a stratified microbial structure. The HFB, HAB, and MA were located in the outer shell, middle layer, and core, respectively. The microbial populations from the bottom compartment were relatively homogeneous in the granular sludge, and from the middle and upper compartments, they were relatively heterogeneous in the granular sludge. The microbial consortia and their spatial distribution were in accordance with the organic loading rate and chemical components in the three compartments.