Toxoplasma gondii, an opportunistic protozoan parasite, infects almost all warm-blooded animals. In this study, we examined the sequence variation in rhoptry protein 20 (ROP20) genes among 18 T. gondii isolates collected from different hosts and geographical regions. Full length ROP20 genes were amplified and sequenced. The results showed that the genes were 1659 bp in length and contained only a single exon, and that the A+T content varied from 46.68 to 47.20% among the 18 strains. The results of sequence alignment indicated that there were 30 variable nucleotide positions (0-1.40%) in the 18 T. gondii strains containing 18 transitions and 11 transversions, representing 1.81% overall sequence variation. Phylogenetic analysis of the ROP20 sequences showed that ROP20 variation could differentiate between the clonal lineage genotypes I and ToxoDB #9, indicating that ROP20 exhibits a relatively marked degree of sequence diversity and might represent a novel genetic marker for intraspecies phylogenetic analyses of T. gondii.