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Were the first ascomycetes yeast-like (unicellular) or filamentous with complex multicellular growth? Molecular studies have indicated that the earliest lineages of ascomycetes were mostly yeast-like and without complex, multicellular fruit bodies. The genus Neolecta stands out as an exception. Neolecta spp. have filamentous growth and discomycete-type of fruit bodies, but constitute one of the basal lineages both in rRNA and RPB2 gene trees. In this paper, we have used 10 previously unpublished and 30 GenBank β-tubulin genes to test the phylogenetic position of Neolecta spp. We compared the phylogenetic information from the amino acid sequences (485 characters) with the phylogenetic information content of 33 characters from intron gains and losses. Due to gene duplications, two paralogous versions of β-tubulin genes occurred in four species included in our analysis. Although phylogenetic interpretation of β-tubulin gene trees was complicated by a history of gene duplications, intron gains and losses, and by unequal rates of amino acid substitution, Neolecta species never formed a monophyletic group with any of the sequences from the filamentous ascomycetes in the Pezizomycotina (euascomycetes) in parsimony or distance analyses. Like the RPB2 and rRNA genes, the β-tubulin genes support the hypotheses of an early divergence of Neolecta from superficially similar filamentous ascomycetes. Neolecta could become a key taxon particularly in comparative studies between the fungal model organisms in the mainly unicellular (=yeast-like) taxa Saccharomyces and Schizosaccharomyces and the filamentous fruit-body forming taxa Neurospora and Aspergillus.