Respiratory syncytial virus (RSV) is the most commonly identified viral agent of acute respiratory tract infection (ARI) of young children and causes repeat infections throughout life. Limited data are available on the molecular epidemiology of RSV from developing countries, including India. This study reports on the genetic variability in the glycoprotein G gene among RSV isolates from India. Reverse transcription-PCR for a region of the RSV G protein gene was done with nasopharyngeal aspirates (NPAs) collected in a prospective longitudinal study in two rural villages near Delhi and from children with ARI seen in an urban hospital. Nucleotide sequence comparisons among 48 samples demonstrated a higher prevalence of group A (77%) than group B (23%) RSV isolates. The level of genetic variability was higher among the group A viruses (up to 14%) than among the group B viruses (up to 2%). Phylogenetic analysis revealed that both the GA2 and GA5 group A RSV genotypes were prevalent during the 2002-2003 season and that genotype GA5 was predominant in the 2003-2004 season, whereas during the 2004-2005 season both genotype GA5 and genotype BA, a newly identified group B genotype, cocirculated in almost equal proportions. Comparison of the nonsynonymous mutation-to-synonymous mutation ratios (dN/dS) revealed greater evidence for selective pressure between the GA2 and GA5 genotypes (dN/dS, 1.78) than within the genotypes (dN/dS, 0.69). These are among the first molecular analyses of RSV strains from the second most populous country in the world and will be useful for comparisons to candidate vaccine strains. Copyright © 2006, American Society for Microbiology. All Rights Reserved.