In an attempt to clarify the relationship between fungi classified as Ophiostoma minus, but of different geographic origins and mating systems, sequencing of the 5.8S and ITS 2 rDNA, and beta-tubulin gene was carried out. The beta-tubulin gene was highly informative, supporting the sub-division of O. minus into two groups based on geographic origin. Furthermore, isolates previously classified as O. pseudotsugae were confirmed as being clearly distinct from O. minus. However, sequencing did not reveal any polymorphisms between isolates with homothallic as compared to heterothallic mating systems. This was supported by crosses using methylbenzamidazole-2-yl carbamate nuclear markers which showed that hybridisation between isolates of different mating systems was possible. However, we propose that different mating systems may still signal a divergence of isolates of O. minus.