Antigenic and genetic heterogeneities exist within the two major antigenic groups of respiratory syncytial (RS) virus. We developed a polymerase chain reaction (PCR)-based assay that not only differentiates the two RS virus groups but allows distinctions within groups on the basis of changes in the nucleotide sequences, as revealed by restriction fragment analysis. In this assay, viral RNA served as a template for cDNA synthesis with extension from a synthetic oligonucleotide primer complementary to bases 164 to 186 in the F protein mRNA. For PCR amplification, two group-specific 5' primers were added. The two primers corresponded to the G protein mRNA sequence of group B (bases 10 to 30) or group A (bases 247 to 267) RS virus. Agarose gel electrophoresis readily discriminated the 1.1-kb group B and the 0.9-kb group A virus amplification products. All 47 viruses tested were assigned to the same group by both PCR and monoclonal antibody reaction pattern analysis. Restriction fragment analysis of the amplified DNAs revealed 12 restriction patterns for group A viruses and 7 restriction patterns for group B viruses, while the monoclonal antibody reaction patterns revealed seven patterns for group A viruses and 3 patterns for group B viruses. Most viruses with the same monoclonal antibody reaction patterns had different restriction patterns, and some viruses with the same restriction patterns had different monoclonal antibody reaction patterns. Thus, the results of the PCR assay concurred with the monoclonal antibody reaction pattern analysis for group classification of RS viruses, while the restriction fragment analysis identified greater diversity within groups than was seen with the monoclonal antibody analysis.