Porcine enteroviruses (PEVs) have been recognized as the causative agents of various clinical manifestations such as fertility disorders, neurological defects, and dermal lesions in pigs. Currently, the diagnosis of PEV infection is carried out by virus isolation, which although useful, is labor- and time-intensive. The present investigation describes the development of a reverse transcription-polymerase chain reaction (RT-PCR) assay for the rapid and sensitive detection of PEVs of cytopathic effect groups I (now known as porcine teschoviruses [PTVs]) and II. The assay described not only detects the PTVs and CPE group II of PEVs but also allows them to be differentiated on the basis of the size of the amplification product, using the same set of oligonucleotide primers. The availability of specific and sensitive molecular diagnostic tools such as the RT-PCR assay described herein should facilitate efficient diagnosis of PTV and CPE group II infections in pigs.