The prevalence of Toxoplasma gondii was surveyed by using a nested polymerase chain reaction (PCR) that was targeted to T. gondii B1 gene in German shepherd dogs and stray cats. Sixty-four (46.3%) out of 138 German shepherd dogs and 50 (47.2%) out of 106 stray cats were tested positive by the nested PCR assay, respectively. There was no significant difference in gender or age in German shepherd dogs and stray cats. In the five positive dogs and five positive cats, the nucleotide partial sequence of the T. gondii B1 gene was identified by direct sequence analysis. All the sequences were identical to each other and the corresponding sequence, T. gondii B1 gene (Accession No. AF179871). The results suggest that the prevalence of T. gondii is high, and the nested PCR assay is useful for early detection of T. gondii for asymptomatic dogs and cats.