Morphology, mitochondrial DNA (mtDNA) restriction fragment polymorphisms (RFLPs) and nuclear DNA (nDNA) fingerprinting were used to clarify relationships among the morphologically similar Ophiostoma and Leptographium species associated with mycangia of three Dendroctonus bark beetles (Ophiostoma clavigerum associated with both D. ponderosae and D. jeffreyi, and L. pyrinum associated with D. adjunctus), as well as a closely related nonmycangial bark beetle associate (L. terebrantis). Most isolates of O. clavigerum form long (40-70 μm), septate conidia, while all isolates of L. terebrantis and L. pyrinum form conidia less than 17.0 μm in length. The conidia of L. pyrinum are pyriform, with truncate bases, while the conidia of the other species form only slightly truncate bases. Conidial masses of L. terebrantis are creamy yellow, while the conidial masses of the other species are white. Nuclear DNA fingerprints resulting from probing PstI restrictions with the oligonucleotide probe (CAC)(5) and HaeIII and MspI restrictions of mtDNA, exhibited three major clusters. In the dendrogram developed from mtDNA RFLPs, the L. pyrinum isolates formed one cluster, while the majority of O. clavigerum isolates, including all D. jeffreyi isolates, formed another. A third cluster was composed of all L. terebrantis isolates, as well as several O. clavigerum isolates from D. ponderosae. The inclusion of some O. clavigerum isolates in the L. terebrantis cluster suggests that horizontal transfer of mtDNA has occurred among these fungi. The nDNA dendrogram also exhibited three clusters, and most isolates of L. pyrinum, L. terebrantis and O. clavigerum grouped separately; however, one isolate of O. clavigerum grouped with the L. terebrantis isolates, while one isolate of L. terebrantis grouped with O. clavigerum. No genetic markers were found that distinguished between O. clavigerum associated with D. ponderosae and O. clavigerum associated with D. jeffreyi. Ophiostoma clavigerum might be a recently diverged morphological variant of L. terebrantis, with special adaptations for grazing by young adults of D. jeffreyi and D. ponderosae. The anamorph of O. clavigerum, Graphiocladiella clavigerum, is transferred to Leptographium.