A method for identifying and differentiating between two highly similar sequence variants in the cucumber mosaic virus RNA 5 population is described. The technique is based on the use of different primers with 3′ terminal mismatches for primer extension analysis. Primers varied in their length, number of 3′ mismatches, and sequence context of the mismatch. The results indicate that the size of the primer is critically important to the ability of this method to differentiate between highly similar RNAs. This technique should prove very useful for the genetic analysis of characteristics that are determined by single nucleotide variations in viral RNA genomes.