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Phylogenetic relationships of one of the largest nonlichen-forming ascomycetous groups, the Leotiomycetes, were inferred from genes encoding three rDNA regions (SSU+LSU+5.8S rDNA). A dataset was prepared with rDNA sequences data from 108 isolates, among which we sampled 85 taxa representing four orders and 16 families in the Leotiomycetes. Equally weighted parsimony and Bayesian analyses were performed. Bootstrap proportion and Bayesian posterior probability under the GTR+gamma+iota model were estimated along the branches. Based on our results the Leotiomycetes is relatively well defined as a class and it includes the Cyttariales, Erysiphales, Helotiales, Rhytismatales and two families of uncertain position, Myxotrichaceae and Pseudeurotiaceae. The placements of the Thelebolales and Ascocorticiaceae are not examined and are accepted as tentative in the Leotiomycetes. Our results agree with previous studies to remove the Geoglossaceae, including Geoglossum, Trichoglossum and Sarcoleotia, from the Leotiomycetes. Positions of the Erysiphales and Rhytismatales in the Leotiomycetes are confirmed. The Helotiales and Myxotrichaceae are paraphyletic. Close relationships are supported strongly among the Hemiphacidiaceae, Rutstroemiaceae and Sclerotiniaceae, among Loramycetaceae, the northern hemisphere Vibrisseaceae, the Dark Septate Endophyte fungus Phialocephala fortinii and Mollisia cinerea, and between species of Bulgaria and Phadidiopycnis. Sequence data of rDNA regions are not adequate to resolve the relationships among major groups of the Leotiomycetes.