During microbial reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), trans-1, 2-dichloroethene (trans-DCE) has been observed to be produced predominantly by certain mixed and pure cultures. However, the reductive dehalogenase (RDase) genes involved in trans-DCE generation remain elusive. In this study, identification and transcriptional analysis of RDases were conducted on trans-DCE-producing Dehalococcoides sp. strain MB. Two pairs of degenerate primers targeting the conserved regions of RDases in known Dehalococcoides species were applied to amplify the putative RDase genes of strain MB. Cloning and restriction analysis revealed the presence of seven unique RDase gene fragments (dceA1 to dceA7) that possess sequence identity to known RDase genes. Gene expression analysis of the PCE-grown culture MB exhibited 10-fold regulation of the RDase gene dceA6 (designated mbrA gene), suggesting that it is involved in the production of trans-DCE. This is in agreement with the molecular size of the most abundant protein that is resolved on the denaturing protein gel. Complete sequence of the mbrA gene was obtained by chromosome walking, and the upstream of it is a regulator of transcription, indicating that the expression of this functional gene is tightly controlled in the microbe. The mbrA gene was subsequently found to be present in other trans-DCE-producing cultures containing Dehalococcoides sp. The new mbrA gene identified in this study may serve as an important biomarker for evaluating, predicting and elucidating the biological production of trans-DCE in the chloroethene-contaminated sites.