Significance: Polychlorinated biphenyls (PCBs) as persistent organic pollutants are widespread in the sediments of lakes, rivers, and harbors. Although the PCB detoxification through microbial reductive dechlorination has been extensively studied for more than 20 y, the difficulty in cultivating PCB dechlorinators in pure culture impedes further characterization, optimization, and application in in situ bioremediation. By combining traditional culture techniques with next-generation sequencing technology, this study reports the successful cultivation and characterization of three PCB-respiring Dehalococcoides mccartyi strains in pure culture and identification of their key functional genes, which advances the PCB bioremediation and our understanding of organohalide respiration of PCBs.