Objective To develop a duplex PCR assay for detection of rat parvovirus H-1 and KRV and its application. Methods To design specific primers on the basis of H-1(NC_001358) and KRV(U790330) genome sequences published in NCBI and establish a duplex PCR assay using H-1 and KRV DNA as templates. To verify the sensitivity and specificity of the method after optimizing PCR. The rats were infected by oral inoculation. The rats were divided into three groups: H-1 infection, KRV infection and mixed infection groups. To collect feces at the 4th, 6th, 8th and 10th days postinfection. Rats were euthanized on the 10th day and samples from heart, liver, spleen, lung, kidney and cecal contents were collected from each rat, then all the samples were screened with the duplex PCR. Results The 183 bp and 302 bp bands were amplified using H-1 and KRV as templates. The sensitivity test showed that the PCR method can detect as low as 3.8 pg/mL H-1 and 0.73 pg/mL KRV. There were no bands amplified when mouse minus virus, canine parvovirus and feline parvovirus were used as templates, showing that the specificity of the duplex PCR assay is very good. The nucleic acids of H-1 or KRV were detected in all rat feces on the 2th day postinfection and there was no obvious clinical symptoms in all the infected rats. The positive rates of H-1 were as follows: 50% (4/8) heart tissues, 50% (4/8) liver tissues, 62.5% (5/8) spleen tissues, 50% (4/8) lung tissues, 37.5% (3/8) kidney tissues and 62.5% (5/8) cecum contents, and the positive rate of single infection group was higher than that of mixed infection group. The positive rates of KRV were as follows: 0 (0/8) heart tissues, 25% (2/8) liver tissues, 87.5% (7/8) spleen tissues, 12.5% (1/8) lung tissues, 25% (2/8) kidney tissues and 62.5%(5/8) cecum contents, and the positive rate of mixed infection group was higher than that of single infection group. Conclusions The duplex PCR assay for H-1 and KRV established in this study can effectively detect H-1 or KRV infection in rat feces and other tissues, and can be used as an effective supplement to the national standard of lab animals.