This study was performed to evaluate the prevalence rate of Toxoplasma gondii on 217 stray cats in Daejeon. The positive infection rate of T. gondii was 15.7% in enzyme-linked immunosorbent assay (ELISA), 12.4% in latex agglutination test (LAT), 14.7% in indirect immunofluorescent antibody test (IFA) and 0.5% in polymerase chain reaction (PCR) respectively. In districts, Yuseong-gu was shown the highest seropositive rate of T. gondii as 31.8% in ELISA, 22.7% in LAT and 31.8% in IFA. In gender, the seropositive rate of female cats was slightly higher than that of male cats as 17.2% in ELISA, 15.2% in LAT, 15.2% in IFA and 1.0% in PCR. Cats captured in National science museum, detached house and apartment was shown relatively high prevalence rate of T. gondii.