Giant cell tumor of bone (GCTB) is a locally aggressive subarticular tumor. Having recently reported that H3.3 G34W mutations are characteristic of this tumor type, we have now investigated the sensitivity and specificity of the anti-histone H3.3 G34W rabbit monoclonal antibody in a wide variety of tumors including histologic mimics of GCTB to assess its value as a diagnostic marker. We also determined the incidence of H3.3 G34 mutations in primary malignant bone tumors as assessed by genotype and H3.3 G34W immunostaining. A total of 3163 tumors were tested. Totally, 213/235 GCTB (90.6%) showed nuclear H3.3 p.G34W immunoreactivity. This was not the case for the rare variants, p.G34L, M, and V, which occurred most commonly in the small bones of the hands, patella, and the axial skeleton. If these sites were excluded from the analysis, H3.3 G34W expression was found in 97.8% of GCTB. Malignant bone tumors initially classified as osteosarcomas were the only other lesions (n=11) that showed G34W expression. Notably an additional 2 previously reported osteosarcomas with a p.G34R mutation were not immunoreactive for the antibody. A total of 11/13 of these malignant H3.3-mutant tumors exhibited an osteoclast-rich component: when imaging was available all but one presented at a subarticular site. We propose that subarticular primary malignant bone sarcoma with H3.3 mutations represent true malignant GCTB, even in the absence of a benign GCTB component.