Technical Abstract: Molecular characterization of Toxoplasma gondii isolates is central f6r understanding differences in disease transmission and manifestations. Despite extreme abundance and vast geographical range, very low genetic diversity has been measured among T gondii isolates. Only three sub-groups (lineages) have been discerned with subtle within-lineage variation, permitting low resolution classification of isolates. Since proteins, coding sequences, and especially antigen-coding genes have been used extensively in previous studies, we focused on sequence variation in introns of house-keeping genes which may be more informative for phylogenetic analysis because they evolve under lower selection. We compared sequence variation in introns of five house-keeping genes with two antigen-coding genes. Low intra-lineage variation was detected in introns of house-keeping genes but not in antigen-coding genes and overall the former were slightly more polymorphic than coding and non-coding regions o antigen-coding genes. Intra-genic linkage disequilibrium was complete, but inter-genic linkage, while being highly significant was incomplete even between genes mapped close to each other, suggesting that genes are partially uncoupled. Six of seven substitutions found within the region coding for the tachyzoite surface antigen, SAG2 were nonsynonymous, indicating that diversifying selection acts on this locus. Typing isolates based on house-keeping and antigen coding genes was consistent, but substantial differences were found in phylogenetic relationships between the resulting groups. A cougar isolate typed as lineage H using an RFLP assay possessed multiple unique polymorphisms, suggesting that it represents a new lineage. We concluded that introns of house-keeping genes