A polymerase chain reaction-based protocol was developed for determination of the sequence of 1300 nucleotides in the E1 coding region of the genomes of multiple strains of rubella virus. From a collection of sequences of 9 independent strains and isolates, characteristic nucleotides were identified that distinguished the RA 27/3 strain, which is the currently used attenuated vaccine strain. These characteristic nucleotides were maintained in virus recovered from recent vaccinees. Both the assay and the knowledge of these characteristic nucleotides should be of use in determining the origin of virus isolated from individuals who have suspected vaccine-associated complications. The nucleotide sequences of the independent rubella virus strains differed by 0.7%-3.6%. Phylogenetic tree analysis of the sequences indicated the existence of at least three distinct genetic lineages.