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The molecular diversity of 14 isolates of Pea seed-borne mosaic virus (PSbMV) from southern Australia, 13 previously described isolates from Pakistan, and a reference isolate from the United States have been studied to determine whether a relatively simple molecular diagnostic assay and classification scheme could be developed for this virus. The Australian isolates were placed into either pathotype P1 or pathotype P4 by bioassay on differential genotypes of Pisum sativum. The Pakistani isolates represented pathotypes P1, P4, U1, and U2, and an undetermined pathotype. The reference US isolate was pathotype P1. A reverse transcription-polymerase chain reaction (RT-PCR) assay based on an amplicon from the variable HC-Pro coding region of polyviruses was shown to distinguish PSbMV from seven other legume infecting polyviruses. Restriction fragment length polymorphisms (RFLPs) generated from the HC-Pro RT-PCR products of all 28 isolates using seven restriction endonucleases placed them into eight groups. A phylogenetic tree based on a Bray-Curtis similarity comparison placed the groups into three clusters. The groups and clusters had no clear association with either pathotype or geographic source. It is concluded that within the range of viruses and isolates tested, the RT-PCR-RFLP method will both specifically identify PSbMV and provide a simple, qualitative, and rapid means for placing PSbMV isolates into groups. Applications could include mapping and tracking isolates in space and time. [ABSTRACT FROM AUTHOR]