A multiplex one-step RT-PCR aiming at detecting all pospiviroids known to be harmful to cultivated plants has been developed. Specificity, sensitivity, selectivity, repeatability and reproducibility of this test have been assessed in order to fulfill the recommendations of the EPPO standard PM7/98 and provide routine detection laboratories with a cost-effective, easy-to-use and robust pospiviroid detection test. To further understand the epidemiology and ease the management of pospiviroid outbreaks, this RT-PCR diagnostic test can be followed by direct sequencing of the amplicons to identify and characterize the detected pospiviroid isolates.