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Toxoplasma gondii typically causes lifelong chronic infection and has been identified in a variety of intermediate and definitive hosts. Felids are capable of serving as both intermediate and definite hosts for T. gondii infection. However, there is no direct evidence to prove that servals are the intermediate host of T. gondii. In this study, T. gondii antibodies were detected in a serval by a modified agglutination test (titer, 1:200). Viable T. gondii was isolated from the striated muscles of the serval. This strain was further propagated in cell culture and designated as TgServalCHn1. Genetic characterization of DNA derived from cell culture was performed by RFLP‐PCR of 10 markers, as well as polymorphic ROP5 and ROP18 genes. Results showed that this strain of T. gondii belonged to the genotype ToxoDB#20. The ROP5 allele 4 and ROP18 allele 3 suggested that this strain was avirulent, which was further supported by infection in mice. Encephalitis, immune organ necrosis and focal mononuclear cell infiltration in multiple organs were the main pathology characteristics observed in BALB/C mice infected with the TgServalCHn1 strain. To our knowledge, the present study is the first to report the isolation of T. gondii from a serval, which gives direct evidence for servals serving as an intermediate host of T. gondii. The genotyping results revealed the presence of genotype ToxoDB#20 in central China, enriching the scope of the distribution of T. gondii genotypes in Asia. [ABSTRACT FROM AUTHOR]