Biodegradation of polychlorinated biphenyls (PCBs) is an important transformation and detoxification route in the environment. To better understand the influence of PCB congener compositions on dechlorination, sediments from two rivers, Hudson and Grasse, and two PCB mixtures (PCB 5/12, 64/71, 105/114, and 149/153/170 in Mixture 1 and PCB 5/12, 64/71, 82/97/99, and 144/170 in Mixture 2) were used for this microcosm study. The Grasse River sediment microcosms exhibited more extensive dechlorination than the Hudson River sediment microcosms. The extent of dechlorination was predominantly controlled by sediment itself, not by the PCB compositions. Rare ortho dechlorination, targeting mono-ortho PCB congeners was observed in Grasse sediment, indicating a potential for full dechlorination of some PCBs in this sediment. The identified ortho dechlorination pathways were PCB 28 (24-4-CB) to PCB 15 (4-4-CB) and PCB 25 (24-3-CB) to PCB 13(3-4-CB). The relative abundances of Dehalococcoides were much higher in both sediments spiked with PCBs. An apparent increase of Dehalococcoides 16S rRNA genes coincided with the commencement of dechlorination. The dechlorination preferences were identified using a modified data analysis approach focusing on chlorine neighboring conditions. In both sediments, the overall dechlorination preferred meta > para > ortho. Specially, ortho-/double-flanked meta-chlorines were primarily targeted followed by single-/double-flanked para-chlorines.