Toxoplasma gondii is one of the most common protozoa parasites of human and other warm-blooded animals. Cats and wild felidae play crucial roles in the epidemiology of toxoplasmosis. This study was performed to survey the prevalence of T. gondii infection among stray cats in the Seoul. A total of 422 stray cat blood samples were collected from Seoul area. Positive sera for T. gondii were identified in 56 samples (13.3%) exclusively via the latex agglutination test and the detection no antigen particles among seropositive samples by PCR. The overall infection rate of male stray cats (14.4%) presented as higher than that of female cats (10.7%). This study suggested that T. gondii is widespread in stray cats of Seoul area. It is needed to control urban stray cat population and to reduced the risk of zoonotic transmission of toxolasmosis to other animals and humans.