Microsporidia are intracellular parasitic microbes, which were once thought to be among early diverging eukaryotes, mainly because they lack mitochondria. However, this evolutionary scheme has been challenged by protein-based molecular phylogenetic studies, which suggest a more recent origin of the Microsporidia, which clustered Microsporidia with or within ‘true’ fungal lineages. However, most of these studies did not include sequences from basal fungal phyla (Chytridiomycota and Zygomycota), and unrepresentative taxon sampling could have resulted in misleading relationships being suggested. To further investigate the evolutionary origin of Microsporidia relative to Fungi, we performed molecular phylogenetic analyses with newly determined RPB1 (DNA-dependent RNA polymerase II largest subunit) and EF-1α (translation elongation factor I alpha) including sequences from basal fungi. Although the phylogenetic position of Microsporidia in the EF-1α tree still might be misplaced due to the unusually high rate of sequence divergence of the microsporidian EF-1α gene, the phylogenies recovered based on these two protein sequences do not provide strong evidences for a close relationship between Microsporidia and Fungi. Moreover, we have identified within the EF-1α genes a characteristic two amino acid deletion conserved in all fungal sequences currently available, whereas this deletion is absent in microsporidian sequences. These results argue against the view of Microsporidia as highly degenerate fungi, and whether Microsporidia and Fungi are sister taxa remains unresolved.