AbstractWe combined museum collection genomics and morphology to identify new genera and species, and to otherwise revise the taxonomy of the poorly understood Australian longhorn beetles in the subfamily Prioninae. Moreover, we produced a comprehensive DNA reference library for molecular species identification and a first comprehensive molecular phylogeny for all Australian Prioninae, using mitogenomic data. The Prioninae include some of the world's largest beetles, and their long‐lived larvae are major recyclers of solid dead timber, yet these longhorn beetles are rarely seen in nature due to their concealed habits, short adult lifespans and nocturnal habits. Furthermore, many species are difficult to identify using morphological characters. We applied Whole Genome Shotgun (WGS) sequencing to 213 historical museum specimens, representing all morphological types of Australian Prioninae. Complete mitochondrial genomes were obtained for all genera, and at least one mitochondrial protein‐coding gene (PCG) was acquired for an additional 141 specimens of Australian Prioninae. Using molecular species delimitation tools, we identified 48 new species of Australian Prioninae, nearly doubling the number of currently recognized Australian species of Prioninae. We assign generic status to Catypnes Pascoe, 1864 stat.r. and Hermerius Newman, 1844 stat.r., propose a new synonymy for Cnemoplites Newman, 1842 (= Paroplites Lameere, 1903 syn.n.), describe six new genera, including Paulhutchinsonia Jin, de Keyzer & Ślipiński gen.n., Papunya Jin, de Keyzer & Ślipiński gen.n., Dunmorium Jin, de Keyzer & Ślipiński gen.n., Hagrides Jin, de Keyzer & Ślipiński gen.n., Cryptipus Jin, de Keyzer & Ślipiński gen.n., Geoffmonteithia Jin, de Keyzer & Ślipiński gen.n., and revise the tribal composition of the Australian Macrotomini. Moreover, our phylogeny of Prioninae provides valuable new insights into the evolutionary origins and nearest relatives of several Australian endemic taxa. Our results emphasize the importance of natural history museums as a repository for information about biodiversity, and highlight the opportunities associated with using museum specimens as a source for molecular data in phylogenetic studies.