Since the emergence of highly pathogenic North American Porcine Reproductive and Respiratory Syndrome virus (H-US-PRRSV) in 2006, the classical North American PRRSV (C-US-PRRSV) and H-US-PRRSV isolates have coexisted in Chinese swine herds. A duplex real-time RT-PCR assay using minor groove binder (MGB) probes for differential detection of the two US PRRSV isolates was developed. The specificity, sensitivity, reproducibility, and interference test of this assay were validated. The sensitivity of the assay was 3.2TCID(50)/ml or 38 RNA copies/microl for C-US-PRRSV and 0.4 TCID(50)/ml or 14 RNA copies/microl for H-US-PRRSV. Both assays were 10 times more sensitive than the current methods. A total of 302 clinical samples were tested by duplex real-time RT-PCR and conventional RT-PCR assays, and the results showed over 98.7% agreement. In addition, the new assay can be completed in less than 2h. This duplex real-time RT-PCR assay is a promising tool for rapid differential detection and epidemiology of US PRRSV in China.