Cognate genes of members of the Palyam serogroup of orbiviruses have been identified previously, and their relatedness to the prototype virus was determined by blot hybridization of the genome segments of members of the serogroup using Palyam genomic RNA and isolated Palyam RNA segments as probes. In this study, the genetic relatedness of nine Palyam serogroup isolates was determined by reciprocal blot hybridizations of genomic RNA from each virus to the segments of all members of the group. The number and identity of highly related genes varied between isolates. For example. CSIRO Village and Palyam were related in genes 2 and 6, while Bunyip Creek and Vellore were related in genes 2 and 6. However, CSIRO Village and Bunyip Creek were highly related to D'Aguilar in all genes except 2 and 6, suggesting that there may have been genetic reassortment of Palyam serogroup dsRNA segments. Genes 2 and 6 were correlated consistently with serotype specificity. Genes 5, 7 and 9 were highly related among all members of the group. The Indian strains, Palyam and Vellore, were highly related in genes 1, 3 and 8, and they exhibited weak homology to genes 1, 3 and 8 of the Australian and African strains. However, one Indian isolate, Kasba, was more closely related to strains from Africa and Australia than it was to other Indian strains. There was little evidence which indicated that geography was predictive of the genetic relationships of the strains. Thus, immunological pressure may be the most important factor affecting the Palyam serogroup gene pool.