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Porcine endogenous retrovirus (PERV) in the pig genome represents a potential infectious risk in xenotransplantation. Chinese miniature pigs have been considered to be potential organ donors in China. However, an adequate level of information on PERV from Chinese miniature pigs has not been available. We established an SYBR Green I-based real-time quantitative polymerase chain reaction (PCR) assay for estimating copy numbers of PERV integrated in the host genome. The assay was 100-fold more sensitive compared with conventional PCR. We also evaluated the specificity and reproducibility of the assay. We statistically analyzed the difference in PERV copy numbers integrated into the genomes of Wuzhishan pigs versus Bama minipigs. This approach will be useful to screen donor pigs as well as to examine clinical samples from human subjects treated with porcine xenotransplantation products for evidence of PERV transmission.