A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the open reading frames 1a of highly pathogenic porcine reproductive and respiratory syndrome virus genome was developed. The 10 reference strains, 1 clinical isolation strain and 122 positive samples were tested. Positive reactions were confirmed for all strains and specimens by reverse transcription loop-mediated isothermal amplification and nested reverse transcription polymerase chain reaction (RT-PCR). The results showed this detection technique is more reliable and convenient for rapid and sensitive diagnosis of highly pathogenic porcine reproductive and respiratory syndrome virus infection.