Lophiostoma and Lophiotrema share several morphological and ecological features. They have been regarded as closely related genera within the family Lophiostomataceae, but their morphological circumscriptions have been uncertain. To clarify the generic definitions of Lophiostoma and Lophiotrema, we conducted phylogenetic analyses of 29 isolates of these genera based on the SSU and LSU nrDNA sequences, and also reevaluated several key characters previously used for their generic characterization. Our results clearly confirmed that Lophiostoma and Lophiotrema are distinct genera belonging to different families; the ascus shape, including length of the ascus stipe, is a reliable taxonomic indicator to allow discrimination between the genera. In Lophiostoma species, asci are clavate with relatively long stipes [mostly (10-) 15-30 lm in length], whereas in Lophiotrema the asci are cylindrical with short stipes (up to 15 lm long). A new family, Lophiotremataceae, is proposed to accommodate species in the Lophiotrema clade that was distantly placed from the Lophiostomataceae within the Pleosporales. Lophiostoma quadrisporum, collected from twigs of Liriodendron tulipifera, is described as a new species with distinctive 4-spored asci. Lophiotrema vitigenum, which has clavate asci with long stipes, is transferred to Lophiostoma.