BACKGROUND Osteomyelitis is a devastating condition whose treatment relies on the detection of bacteria. The current standard of microbiology culture may not be adequate. Molecular biology based diagnostic procedures for detecting bacteria in orthopaedic infections was previously established, but has not been applied to the setting of chronic osteomyelitis. We aim to determine the applicability of molecular diagnostic procedures for monitoring chronic osteomyelitis, and to evaluate if these procedures are superior to standard culture methods of osteomyelitis detection. METHODS A rabbit experimental model of chronic osteomyelitis was used; infection was induced in the proximal, medial aspect of the tibia with Staphylococcus aureus at titers ranging from 1 × 10(2) to 1 × 10(6) colony forming units. At 28 days post-infection, animals were sacrificed, and the tibias were examined radiographically, harvested, and assayed for the presence of bacteria. Two bacterial detection methods were used: (1) standard microbiological culturing, and (2) polymerase chain reaction (PCR) based diagnostic method to detect bacterial genomic DNA. RESULTS The molecular diagnostic method was highly sensitive and accurate, and detected low titer infections that were undetected by radiographic and microbiological methods. By using two sets of PCR primers, one for a universal bacterial gene (16S rRNA) and one for a species-specific gene (nuc), the molecular protocol allowed both the detection and speciation of the bacterial infection. CONCLUSIONS The use of the PCR-based method was effective for high-sensitivity detection and identification of bacteria associated with chronic osteomyelitis in a rabbit model. Our findings illustrate the applicability of PCR for monitoring chronic osteomyelitis, which may be useful for improved detection of osteomyelitis organisms in humans.