Populations of the D-satellite RNA (D-sat) of cucumber mosaic virus (CMV) have a major site of heterogeneity at which a significant proportion of the molecules vary from each other. The generation of this heterogeneity was investigated by using infectious transcript RNA of a prototype D-sat clone as an inoculum of uniform sequence to initiate serial passages in five different host plant species. RNase protection assays indicated that heterogeneity at the same site seen in natural D-sat RNA populations developed within two to four passages in most of the hosts tested, and the proportion of the population exhibiting this heterogeneity accumulated to a much higher level in tobacco than in the other hosts. Characterization of the heterogeneity which developed in tobacco showed it to be qualitatively different from that in the original D-sat population. This analysis of a CMV satellite RNA is the first example in which infectious transcript RNA from a clone has been used to investigate the rapid generation and selection of heterogeneity in a genomic RNA population replicating in plants.