The purpose of this study was to develop a multiplex PCR that can detect porcine endogenous retrovirus (PERV) proviral genes (pol, envA, envB, envC) and porcine mitochondrial DNA, using a dual priming oligonucleotide (DPO) system. The primer specifically detected the PERV proviral genes pol, envA, envB, envC, and porcine mitochondrial DNA only in samples of pig origin. The sensitivity of the primer was demonstrated by simultaneous amplification of all 5 target genes in as little as 10 pg of pig DNA containing PERV proviral genes and mitochondrial DNA. The multiplex PCR, when applied to field samples, simultaneously and successfully amplified PERV proviral genes from liver, blood and hair root samples. Thus, the multiplex PCR developed in the current study using DPO-based primers is a rapid, sensitive and specific assay for the detection and subtyping of PERV proviral genes.