Strain Ochrobactrum lupine TP-D1 was found to degrade chlorothalonil (TPN) to 4-hydroxy-chlorothalonil (TPN-OH). To clone the related degrading gene, genomic library of TP-D1 was constructed using Escherichia coli DH10B and two positive clones 889 and 838 were gained. However, no plasmid was detected in clone 889. And in clone 838, a 3494 bp fragment was cloned which contains a 984 bp hydrolytic dehalogenase (chd) gene and a 1926 bp insertion element IS-Olup. The insertion element contains a transposase coding region (1026 bp), an ATP-binding protein coding region (657 bp) and flanked by 20 bp inverted repeat sequences. Further isolation provided another seven TPN-degrading strains, they belonged to the genera of Pseudomonas sp., Achromobacter sp., Ochrobactrum sp., Ralstonia sp., and Lysobacter sp. PCR strategy showed that they all contain the same structure of chd gene and the upstream IS-Olup. Our evidences collectively suggest that chd gene may be disseminated through horizontal gene transfer based on phylogenetic analysis of the cluster and their host bacterial strains. At the same time, the chd gene was amplified from genome of the positive clone 889, which also provides some potential evidence to the gene horizontal transfer.