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ABSTRACT. The intracellular protozoan Toxoplasma gondii is one of the most successful parasites, with the ability to invade all warmblooded animals, including humans. T. gondii heat shock protein 60 (TgHSP60) plays an important role in intracellular survival and in the differentiation of the parasite, and is also recognized as being associated with its virulence. In the present study, we examined sequence variation in the hsp60 coding region among five T. gondii isolates from different hosts and geographical regions, which were compared with the corresponding sequences of strains ME49, 76K, and GT1 available in the ToxoDB databases. The length of the T. gondii hsp60 sequence was 1728 bp for all strains, and the A+T content ranged from 41.96 to 42.13%. The sequence alignment of the 8 T. gondii strains identified 20 variable positions (0-1.44%) and showed 1.16% overall sequence 6907 ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 13 (3): 6906-6911 (2014) Sequence variation in the T. gondii hsp60 gene variation, suggesting a relatively considerable sequence diversity. Phylogenetic analysis of hsp60 sequences using Bayesian inference and maximum parsimony differentiated the two major clonal lineage types into their respective clusters, and thus separated atypical strains from classical genotypes. The results of the present study suggested that the coding region of the hsp60 gene may represent a novel genetic marker for intraspecies phylogenetic analyses of T. gondii.