Eleven cultures representing eight genera of five families of Loculoascomycetes were obtained from ATCC and IMI. Partial 18S and 28S nuclear ribosomal DNA (rDNA) were amplified using PCR and sequenced. About 1100 bps of 18S rDNA (NS1/NS4 region) and 459 bps of 28S rDNA (F63/R635 region) for each taxon were subject to phylogenetic analysis using parsimony. The reconstructed phylogenetic trees supported the separation of two monophyletic groups, containing the type genera of the Leptosphaeriaceae and the Pleosporaceae, respectively. The members of the Leptosphaeriaceae (sensu Barr) consisted of the paraphyletic clade in the phylogenetic trees. The members of the Pleosporaceae (sensu Barr) plus Pyrenophora trichostoma formed a monophyletic clade. The results generally supported the separation of the Leptosphaeriaceae from Pleosporaceae and the placement of P. trichostoma, and Comoclathris baccata in the Pleosporaceae. The taxonomic position of Pleospora betae, whose coelomycetous anamorph is more closely related to Leptosphaeriaceae, was not well resolved because of low bootstrap values and decay indices. Leptosphaeria bicolor, whose ascospore morphology is not congeneric with the type of the genus, L. doliolum, does not belong to Leptosphaeria based on the 18S sequence data. The taxonomic disposition of Lewia, Pyrenophora, and Comoclathris based on the phylogenetic analyses are discussed.