The objective of the present study was to determine the characterization of Toxoplasma gondii in cats, rats, and chickens in the border areas of Yunnan Province. A total of 259 samples was collected from 10 border areas in Yunnan Province including 94 cats, 58 rats, and 107 chickens. Samples were screened by a nested polymerase chain reaction (PCR) assay and the positive products were analyzed by multilocus PCR-restriction fragment length polymorphism (RFLP) to determine the genotypes. Toxoplasma gondii deoxyribonucleic acid (DNA) was detected from 15.96% of 94 cats, 15.52% of 58 rats, and 6.54% of 107 chickens, respectively, and the average infection rate is 11.97%. Using the multilocus PCR-RFLP, we found that the genotype of T. gondii in cats and rats was ToxoDB#9. Because of low DNA concentration, no genotype was determined from chickens. These results fill the gaps of knowledge in the prevalence and genotype of T. gondii in the border areas of Yunnan Province and have implications for the better control of T. gondii infection in humans and animals.