Though the diagnostic utility of H3.3G34W immunohistochemistry (IHC) for giant cell tumor of bone (GCTB) is well established, it is limited by a proportion of cases with variant H3-3A mutations [H3-3A: c.104G>T p. Gly35Val (G34V), H3-3A: c.103G>A p. Gly35Arg (G34R), H3-3A: c.103_104delinsCT p. Gly35Leu (G34L)], wild-type genotype and H3-3A:c.103G>T p. Gly35Trp (G34W) mutation identified by sequencing but not by IHC. In this study, the H3-3A gene mutation by Sanger sequencing was analyzed in a large cohort of GCTB and its histological mimics. Sequencing of the H3-3A gene was performed to detect mutations in 148 GCTBs and 57 histologic mimics. The other osteoclast giant cell containing lesions histologically mimicking GCTB included were chondroblastoma (22), aneurysmal bone cyst (11), chondromyxoid fibroma (6), telangiectatic osteosarcoma (6), brown tumor of hyperparathyroidism (4), clear cell chondrosarcoma (3), osteoid osteoma (2), osteoblastoma (2) and giant cell reparative granuloma (1). Of the 148 GCTBs tested, 129 showed H3-3A gene mutations by Sanger sequencing and remaining 19 were wild type. The different H3-3A mutations detected included 126 H3-3A:c.103G>T p. Gly35Trp (G34W), one each of H3-3A: c.103G>A p. Gly35Arg (G34R), H3-3A: c.104G>T p. Gly35Val (G34V) and H3-3A: c.103_104delinsCT p. Gly35Leu (G34L). The results of H3-3A gene sequencing were in concordance with the immunohistochemical expression for H3.3G34W/R/V in 129 tumors. All the 57 osteoclast giant cell-containing lesions, other than GCTB, except one (1) case of chondroblastoma, did not show any mutations in the H3-3A gene. The latter case showed H3-3A: c.110A>T p. Lys37Met (K36M) mutation. The H3-3A gene sequencing assay demonstrated a sensitivity of 87.16% and an absolute specificity of 100% among the cases analyzed in the study. Determination of the H3-3A gene mutation by sequencing is a highly sensitive and absolutely specific diagnostic tool for the diagnosis of GCTB and differentiation from its histologic mimics.