The molecular pathogenesis of mixed odontogenic tumors has not been established, and understanding their genetic basis could refine their classification and help define molecular markers for diagnostic purposes. Potentially pathogenic mutations in the component tissues of 28 cases of mixed odontogenic tumors were assessed. Laser capture microdissected tissue from 10 ameloblastic fibromas (AF), 4 ameloblastic fibrodentinomas (AFD), 6 ameloblastic fibro-odontomas (AFO), 3 ameloblastic fibrosarcomas (AFS), and 5 odontomas (OD) were screened by next-generation sequencing and results confirmed by TaqMan allele-specific quantitative PCR. BRAF p.V600E mutation in the mesenchymal component was shown in 4 of 10 AF (40%), 2 of 4 AFD (50%), 2 of 6 AFO (33%), and 2 of 3 AFS (67%), whereas all 5 OD were wild type for BRAF p.V600E. Mutation in the epithelial component was only observed in one AF and one AFO. One AFS contained an area of benign AF, and the mesenchymal component of both (AFS and AF) contained BRAF p.V600E, supporting the concept of malignant progression from a benign AF precursor. KDR, TP53, KIT, and PIK3CA single-nucleotide polymorphisms are reported. In conclusion, AF, AFD, AFO, and AFS show BRAF p.V600E in their mesenchymal component, unlike OD, which are BRAF wild type, suggesting that at least a subset of AF, AFD, and AFO are molecularly distinct from OD, and may represent distinct entities and be neoplastic.