Recent studies showed that the huge diversity branching at or near the phylogenetic root of the fungal kingdom, mostly constituted by uncultured environmental clones, is actually characterized by intracellular predators/parasites of various eukaryotes. These form three related lineages: the Aphelidea, endoparasites of algae; the Rozellomycota, with Rozella species mainly endoparasites of water moulds, and Paramicrosporidium species endonuclear parasites of amoebae; and the Microsporidia, mainly endoparasites of animals. Increasing evidence suggests the emergence of Microsporidia from within Rozellomycota; however, their fungal or protistan nature is still unclear. Here, we report the molecular phylogeny based on the small subunit ribosomal RNA (SSU rDNA) gene, of an additional endoparasite of amoebae, corresponding to the old enigmatic chytrid Nucleophaga amoebae described in the nineteenth century. Our results show that Nucleophaga, possessing a morphotype intermediate between Rozella and Paramicrosporidium, emerges as a unique lineage within the Rozellomycota. The recovery and characterization of new members of Rozellomycota are of high value for the understanding of the early evolutionary history of the Fungi and related lineages.