Burkholderia cepacia is a problematic pathogen that may spread among patients with cystic fibrosis (CF). One highly infectious CF strain that causes epidemics in both the United Kingdom and eastern Canada has been shown to possess both the cable pilin subunit gene (cblA) and a unique combination of insertion sequences. However, no genetic markers linking this strain type with other types epidemic at various centers have been identified. Using a randomly amplified polymorphic DNA (RAPD) typing scheme, we identified an apparently conserved 1.4-kb fragment in the DNA fingerprint of epidemic B. cepacia strains. Conservation of the DNA marker among epidemic strains was demonstrated by Southern hybridization, and its prevalence was assessed in a collection of chromosomal DNAs extracted from 627 isolates representative of 132 RAPD-defined B. cepacia strain types. The marker was specifically associated with seven epidemic CF strains, was absent among nonepidemic strains infecting individual patients with CF, and rare among strains recovered from the natural environment. Only one of the seven epidemic CF strain types possessed DNA homologous to cblA. The RAPD marker was designated the "B. cepacia epidemic strain marker" (BCESM). Sequence analysis of chromosomal DNA corresponding to the 1.4-kb RAPD marker revealed the presence of a putative open reading frame (ORF) with significant homology to several negative transcriptional regulators; the ORF was designated the "epidemic strain marker regulator," or esmR. The BCESM DNA is the first genetic marker that has been identified to be specifically associated with and conserved among several epidemic B. cepacia strains which infect multiple patients with CF.