Cladistic analysis of characters derived from nuclear-encoded 18S rDNA sequences was used to infer the phylogenetic placement of ambrosial species of the anamorph genus Raffaelea among perithecial ascomycetes. Of eight species in the genus investigated, seven resolve as a monophyletic lineage that forms a sister group to the sexual genus Ophiostoma. Raffaelea hennebertii, the single species excluded from this lineage, appears to be allied phylogenetically with species of Melanospora. These data are discussed in relation to the monophyly of Raffaelea and its relationship with other sexual and asexual fungal associates of wood-boring Coleoptera. In addition, we report the presence of significant length variation in PCR-amplified 18S rDNA fragments for three of the Raffaelea species studied.