The prevalence of anti-Toxoplasma gondii specific IgG in stray and household cats in Guangzhou, China was determined by ELISA on serum samples from 206 cats (117 strays and 89 households) and the overall infection rate was 25.24%. The infection rate in stray cats (30.77%) was significantly higher (P < 0.05) than in household cats (17.98%). The rate of infection between male and female cats of both groups was not significantly different (P ≥ 0.05), 28.13% versus 32.61% for male and female in stray cats, respectively, and 18% versus 17.95% in household cats. The present investigation demonstrated that the prevalence of T. gondii infection in cats in Guangzhou was high, especially in stray cats, which are probably the main source of T. gondii infection in this area. Integrated control strategies and measures should be implemented to prevent and control T. gondii infection in both stray and household cats, which will have significant implications for the control of human infection with T. gondii.