To develop robust and reproducible methods for molecular typing of <I>Toxoplasma </I>strains, the DNA regions of 5<I>S</I> rDNA, 28S-18S rDNA <I>IGS SAG2</I>, and <I>GRA6</I> loci were examined. The 5<I>S</I> sequences were identical among 24 different strains; sequencing of the <I>IGS</I> region showed a few polymorphisms (0.66%) distinguishing virulence types. The IGS PCR-RFLP methods were developed and used to examine 29 strains of different virulence types. Sequence analysis of the IGS 5'-end showed great diversity between <I>Neospora caninum </I>and <I>T. gondii. </I>The IGS-RFLPs also clearly distinguished between those two closely related species. Nucleotide sequencing of the <I>SAG2</I> locus (a surface antigen coding gene) showed 1.37% polymorphisms among 24 strains. Apart from a single nucleotide change at the 5'-flanking region, the type III and type I strains were identical. However, three new alleles of this locus were identified in minor variants of the strains. Analysis of the coding region of the <I>GRA6</I> locus (a dense granule antigen coding gene) revealed a great degree of polymorphisms (3.24%) among 33 strains. Nine different alleles, representing the three current types and the minor variants of strains were characterised at this locus. A PCR-RFLP based on <I>GRA6</I> polymorphisms was developed which could distinguish the three major types of <I>T. gondii</I>. This marker proved to be a suitable tool for a population study of the <I>Toxoplasma </I>parasite. The predominance of non-synonymous nucleotide substitutions in <I>SAG2</I> and <I>GRA6</I> genes confirmed positive selection in these loci, suggesting they play an important role in the parasite virulence. Phylogenetic analysis based on the multi-locus sequence alignment showed the existence of more than three lineages in <I>Toxoplasma </I>populations.