Toxoplasmosis is caused by the intracellular protozoan Toxoplasma gondii. It is anopportunistic zoonosis in warm-blooded animals and humans, with a worldwide distribution. Toxoplasma gondii dense granule protein 16 (TgGRA16) can modulate some functions in host cells and is considered a significant virulent factor of the parasite. The present study reports sequence variation in TgGRA16 gene among T. gondii strains from different hosts and geographical locations, and the construction of phylogenetic relationships of these T. gondii strains based on sequences of TgGRA16, and analysis of B cell epitopes in TgGRA16. Our results showed that all TgGRA16 gene sequences were 1518 bp and the C+G contents ranged from 52.17% to 52.59%. Sequence variation in the TgGRA16 gene was 0-1.51%. Phylogenetic analysis revealed that TgGRA16 gene sequence could not be used to differentiate the different T. gondii genotypes. Six B cell epitopes were predicted in TgGRA16. These results indicated that TgGRA16 gene is not an ideal marker for studying genetic relationships of T. gondii isolates, but may represent a good vaccine candidate against toxoplasmosis.