A promising genetic marker, sag5b, was cloned and expressed and the difference of the genes between highly virulent strain (RH) and less virulent strain(Prugniaud) of Toxoplasma gondii was compared. The PCR-generated product of sag5b was subcloned into T easy vector and plasmid pET28a consecutively. The fusion expression was induced by IPTG and identified by SDS-PAGE and Western blotting. The immunoreactivity of recombinant SAG5B was identical to that of native SAG5B on the membrane of tachyzoites of RH strain. The brains of mice infected with Prugniaud strain of T. gondii were homogenated. Sag1 was successully cloned by PCR from both RH strain tachyzoites and the homogenized brain tissues of mice infected with low virulent strain of Prugniaud,whereas sag5b was only detected in RH strain but not in Prugniaud strain, indicating that sag5b could be used as a genetic marker for differentiation of strain virulence. Expression and vaccination of the virulence-associated gene into mice failed to induce obvious protective immunity against the challenge of RH strain.