Phylogenetic lineages of pathogenic Streptococcus agalactiae (group B streptococci [GBS]) can be identified by analysis of restriction-digestion patterns (RDPs) of chromosomal DNA. The purpose of the present study was to correlate GBS RDP types and (1) alleles of the highly conserved gene encoding translation-initiation factor IF2, infB, and/or (2) the inserted elements IS1548 and GBSi1. Only 1 combination of serotype and infB allele was found within each RDP type. Strains within a particular RDP type also tend to have the same inserted elements in each of 3 loci examined. A novel insertion sequence, designated "IS1563," was found within all RDP type II-2 strains. Most RDP types could be identified by a combination of serotype, infB allele, and inserted elements at each of the loci. These molecular markers can be used to identify GBS populations and to correlate RDP types and phylogenetic lineages identified by different methods.