Prognosis of chronic osteomyelitis depends heavily on proper identification and treatment of the bone-infecting organism. Current knowledge on selecting the best specimen for culture is confusing, and many consider that non-bone specimens are suitable to replace bone cultures. This paper compares the microbiology of non-bone specimens with bone cultures, taking the last as the diagnostic gold standard.Retrospective observational analysis of 50 patients with bacterial chronic osteomyelitis in a 750-bed University-based hospital.Concordance between both specimens for all etiologic agents was 28%, for Staphylococcus aureus 38%, and for organisms other than S. aureus 19%. The culture of non-bone specimens to identify the causative organisms in chronic osteomyelitis produced 52% false negatives and 36% false positives when compared against bone cultures.Diagnosis and therapy of chronic osteomyelitis cannot be guided by cultures of non-bone specimens because their microbiology is substantially different to the microbiology of the bone.