A splenectomized patient with hypogammaglobulinemia who was hospitalized because of a high-grade fever subsequently developed osteomyelitis. Although pus cultures were repeatedly sterile, polymerase chain reaction (PCR) analysis with use of 16S rRNA gene primers with a broad specificity detected bacterial DNA in pus samples. Subsequent nucleotide base determination of the amplified DNA demonstrated that the detected DNA was derived from Mycoplasma pneumoniae. The results were confirmed by a PCR assay with use of M. pneumoniae-specific primers. Our findings confirm the usefulness of 16S rRNA gene amplification and analysis in the rapid and specific diagnosis of infectious osteomyelitis and reaffirm the role of such methods in detecting fastidious or uncultivable pathogens.