To compare the incidence of osteomyelitis based on different operational definitions using the gold standard of bone biopsy, we prospectively enrolled 35 consecutive patients who met the criteria of ≥21 years of age and a moderate or severe infection based on the Infectious Diseases Society of America classification. Bone samples were obtained from all patients either by percutaneous bone biopsy, or by intraoperative culture if the patient required surgery. Bone samples were analyzed for conventional culture, histology, and 16S rRNA genetic sequencing. We evaluated five definitions for osteomyelitis: 1. traditional culture 2. histology 3. genetic sequencing 4. traditional culture and histology 5. genetic sequencing and histology. There was variability in the incidence of osteomyelitis based on the diagnostic criteria. Traditional cultures identified more cases of osteomyelitis than histology (68.6% vs. 45.7%, p=0.06, OR 2.59, 95% CI 0.98-6.87) but not significant. In every case that histology reported osteomyelitis, bone culture was positive using traditional culture or genetic sequencing. 16S rRNA testing identified significantly more cases of osteomyelitis compared to histology (82.9% vs. 45.7%, p=0.002, OR 5.74, 95% CI 1.91-17.28) and more compared to traditional cultures but not significant (82.9% vs. 68.6%, p=0.17, OR 2.22, 95% CI 0.71-6.87). When both histology and traditional culture (68.6%) or histology and genetic sequencing cultures (82.9%) were used to define osteomyelitis, the incidence of osteomyelitis did not change. There is variability in the incidence of osteomyelitis based on how the gold standard of bone biopsy is defined in diabetic foot infections.