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Toxoplasma gondii, an obligate intracellular parasite, is able to infect many animal species and humans, and can cause toxoplasmosis of the host. In this study, we examined sequence variation in rhoptry protein 47 (ROP47) gene among T. gondii isolates originating from different hosts and geographical regions. The entire genome region of the ROP47 gene was amplified and sequenced, and phylogenetic relationship was reconstructed using maximum parsimony (MP), maximum likelihood (ML) and neighbor-joining (NJ), based on the ROP47 gene sequences. The results of sequence alignments showed that all ROP47 gene sequences were 396 bp in length. There were 19 variable nucleotide positions in the coding region, resulted in 16 amino acid substitutions (12.21%) among all examined T. gondii strains and the existence of polymorphic restriction sites for endonucleases SacI and AflIII, allowing the differentiation of the three major clonal lineage types I, II and III by PCR-RFLP. Phylogenetic analysis of ROP47 gene sequences showed that three major clonal lineage types I, II and III were clustered differently, consistent with PCR-RFLP results. These results suggest that ROP47 gene sequence may represent a potential novel genetic marker for population genetic studies of T. gondii isolates., (Copyright © 2015 Elsevier Inc. All rights reserved.)