The diagnosis of malignant peripheral nerve sheath tumor is challenging, particularly in the sporadic setting. Inactivation of the polycomb repressive complex 2 (PRC2), resulting from inactivating mutations of its constituents SUZ12 or EED1, has recently been identified in 70-90% of malignant peripheral nerve sheath tumors. Homozygous PRC2 inactivation results in loss of histone H3K27 trimethylation (H3K27me3). PRC2 inactivation promotes tumor progression and may render patients sensitive to epigenetic-based targeted therapies. H3K27me3 loss has not yet been validated as a diagnostic marker. We evaluated immunohistochemistry for H3K27me3 in 100 malignant peripheral nerve sheath tumors (70 sporadic, 10 neurofibromatosis type 1-associated, 10 radiation-associated, 10 epithelioid) and 200 other spindle cell neoplasms representing potential mimics (20 each monophasic synovial sarcoma, leiomyosarcoma, dedifferentiated liposarcoma, malignant solitary fibrous tumor, low-grade fibromyxoid sarcoma, cellular schwannoma, spindle cell melanoma, unclassified postradiation sarcoma; 10 each atypical neurofibroma, spindle cell rhabdomyosarcoma, gastrointestinal stromal tumor, fibrosarcomatous dermatofibrosarcoma protuberans). In total, 51 (51%) malignant peripheral nerve sheath tumors, including 34 (49%) sporadic, 7 (70%) neurofibromatosis type 1-associated, and 10 (100%) radiation-associated, but no epithelioid malignant peripheral nerve sheath tumors, were negative for H3K27me3. An additional 6 (6%) tumors showed heterogeneous H3K27me3 expression. Among the 90 sporadic, neurofibromatosis type 1-associated, and radiation-associated malignant peripheral nerve sheath tumors, complete H3K27me3 loss was observed in 29% of low-grade, 59% of intermediate-grade, and 83% of high-grade tumors (low vs intermediate/high grade, P=0.0003). Among other tumor types, 4 (20%) unclassified postradiation sarcomas were negative for H3K27me3, whereas all other neoplasms were positive. Loss of H3K27me3 is highly specific for malignant peripheral nerve sheath tumor (although only modestly more sensitive than S-100 protein and SOX10) and may be a useful diagnostic marker. Our findings suggest that PRC2 inactivation in malignant peripheral nerve sheath tumor may occur during progression to higher grades.