The depiction of evolutionary relationships within phylum Ascomycota is still controversial because of unresolved branching orders in the radiation of major taxa. Here we generated a dataset of 166 small subunit (18S) rDNA sequences, representative of all groups of Fungi and used as input in a Bayesian phylogenetic analysis. This phylogeny suggests that Discomycetes are a basal group of filamentous Ascomycetes and probably maintain ancestor characters since their representatives are intermingled among other filamentous fungi. Also, we show that the evolutionary rate heterogeneity within Ascomycota precludes the assumption of a global molecular clock. Accordingly, we used the penalized likelihood method, and for calibration we included a 400 million-year-old Pyrenomycete fossil considering two distinct scenarios found in the literature, one with an estimated date of 1576 Myr for the plant-animal-fungus split and the other with an estimated date of 965 Myr for the animal-fungus split. Our data show that the current classification of the fossil as a Pyrenomycete is not compatible with the second scenario. Estimates under the first scenario are older than dates proposed in previous studies based on small subunit rDNA sequences but support estimates based on multiprotein analysis, suggesting that the radiation of the major Ascomycota groups occurred into the Proterozoic era.