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<p>Abstract</p> <p>Background</p> <p><it>Toxoplasma gondii</it> is a highly prevalent protozoan parasite infecting a wide range of animals and humans. The epidemiological and biological diversity of <it>T. gondii</it> has resulted in a high genetic variation and unusual population structure in this parasite. This study examined sequence diversity in dense granule 5 (GRA5) gene among <it>T. gondii</it> isolates from different hosts and geographical regions.</p> <p>Methods</p> <p>The entire genome region of the GRA5 gene was amplified and sequenced from 14 <it>T. gondii</it> isolates, and phylogenetic relationship among these <it>T. gondii</it> isolates was reconstructed using Bayesian inference (BI) and maximum parsimony (MP) based on the GRA5 sequences.</p> <p>Results</p> <p>The complete sequence of the GRA5 gene was 1614 bp in length for strains TgCatBr5 and MAS, but 1617 bp for the other 12 strains. Sequence analysis identified 41 (0–1.7%) variable nucleotide positions among all isolates, with 18 variations of these being in the coding region. Variable positions in the coding region resulted in 11 amino acid substitutions, and a deletion of 3 bp in the strains TgCatBr5 and MAS leading to the deletion of one amino acid. Sequence variations resulted in the existence of polymorphic restriction sites for endonucleases <it>Aat</it>II and <it>Mlu</it>I, allowing the differentiation of the three major clonal lineage types I, II and III by PCR-RFLP. Phylogenetic analyses using BI and MP supported the clear differentiation of the examined <it>T. gondii</it> strains into their respective genotypes.</p> <p>Conclusions</p> <p>This study demonstrated the existence of sequence variability in the GRA5 gene sequence among <it>T. gondii</it> isolates from different hosts and geographical regions, which allowed the differentiation of the examined <it>T. gondii</it> strains into their respective genotypes, suggesting that this highly polymorphic GRA5 locus may provide a new genetic marker for population genetic studies of <it>T. gondii</it> isolates.</p>