Reverse transcriptase-polymerase chain reaction (RT-PCR) methods, based on the sequences of RNA segments 5, 7 and 9 of Chuzan virus, were established for specific detection and molecular characterization of the Palyam serogroup orbiviruses. Nucleotide sequences obtained from the amplified cDNA fragments of these three genes of 24 isolates were analyzed and compared individually to determine the intra-serogroup phylogenetic relationship of Japanese, Australian and Zimbabwean isolates. It seems that Chuzan virus isolates in Japan are genetically stable. Interestingly, mutations have occurred almost simultaneously on these three genes of Chuzan virus. In all cases, isolates from the same geographical area were closely related to each other at the molecular level, irrespective of serotype. The data suggested that the Palyam serogroup viruses can be differentiated into geographically distinct groups and that the viruses evolve independently in the different gene pools. A strain KY-115 was considered to be produced by reassortment of genome segments between different groups. Restriction fragment length polymorphism (RFLP) analysis of these PCR products is useful for rapid discrimination of isolates and for detection of genetic mutations.