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Toxoplasma gondii, a highly prevalent obligate apicomplexan parasite, is important in clinical and/or veterinary medicine. Based on its virulence in laboratory mouse strains, T. gondii is divided into three lineages. In this study, we examined and compared the distributions of the glycerophospholipids phosphatidylserine (PtdSer) and phosphatidylethanolamine (PtdEtn) and a glycosphingolipid GM3 in type I RH and type II PLK strains of T. gondii (highly and moderately virulent phenotypes, respectively). The gold labeling density of PtdSer in the exoplasmic leaflet (EF) of the inner membrane complex (IMC) in the RH strain was significantly higher than that in the PLK strain. PtdEtn labeling was significantly higher in the EF than in the cytoplasmic leaflet (PF) of the middle and inner IMC membranes in the RH strain. In contrast, PtdEtn labeling was low in the PLK strain. The gold labeling density of GM3, localized in the EF of the IMC membrane, was much higher in the RH strain than that in the PLK strain. GM3 is a major raft component and both PtdSer and PtdEtn affect the maintenance of raft microdomains, which are important for gliding motility when T. gondii enters mammalian host cells. Raft microdomains have significant roles in the virulence of T. gondii. Therefore, we suggest that the PtdSer and PtdEtn expression levels are correlated with the virulent phenotypes of T. gondii. Furthermore, we demonstrated that PtdSer was absent in the PF of the inner IMC membrane but present in the PF of the middle IMC membrane. This suggests that a barrier-like mechanism that prevents the diffusion of PtdSer exists in the PF between these two membranes.