The loculoascomycetes are defined by the initiation of ascoma development prior to dikaryotisation and functionally bitunicate asci. Previous molecular studies suggested non-monophyly of loculoascomycetes and consequently, Chaetothyriomycetes and Dothideomycetes were distinguished. We have used sequences of nu SSU rDNA, nu LSU rDNA, and mt SSU rDNA to re-evaluate the monophyly of loculoascomycetes. 13 new sequences of these regions from 10 species, including two representatives of Pezizomycetes used as outgroup, were aligned with sequences obtained from GenBank. A combined data set was analysed phylogenetically using maximum parsimony. The Chaetothyriomycetes and Eurotiomycetes form a sister-group, supported by a bootstrap value of 97%, suggesting that loculoascomycetes are not monophyletic. A topology constrained to loculoascomycete monophyly can be rejected as being significantly worse using parametric bootstrapping. Our results also indicate that mt SSU rDNA sequence data are useful as additional characters to elucidate the phylogeny of ascomycetes at the rank of different classes. Additional data from more representatives of these groups and other ascomycetes are required to clarify whether the loculoascomycetes are a paraphyletic or polyphyletic assemblage.