Sequence heterogeneity within populations of three satellite RNAs of cucumber mosaic virus (CMV) was assessed using two different approaches. Comparison of the nucleotide sequences of several cDNA clones of each satellite RNA revealed microsequence heterogeneity, which is often seen in populations of RNA genomes. RNase protection assays using minus-sense satellite RNA probes were used to detect major sites of heterogeneity within natural populations of each satellite RNA. In RNase protection assays of WL1-sat RNA populations, no major sites of heterogeneity were detected within seven different populations, including preparations from four different host plant species. In contrast, RNase protection assays of nine populations of B1-sat RNA showed three different patterns, which were most likely due to the existence of the B1-sat RNA populations as mixtures in which different sequence variants predominated in different preparations. Assays of five independent populations of D-sat RNA revealed a single major site of heterogeneity which was common to each population and was localized at approximately nucleotide 225 of the 335-nucleotide satellite sequence. This common site of heterogeneity is a feature of the D-sat RNA population structure which may represent an equilibrium between alternative nucleotides at a selectively neutral position, or may be actively selected for and maintained.