A new amoeba species pathogenic for Senegalese sole is described based on ultrastructural analysis and SSU rDNA phylogenetic inference. The parasite presents round to ovoid trophozoites (<5 μm) with a high degree of intracellular simplification. No mitochondria were observed, but mitosome-like organelles were present. No cysts could be detected. Phylogenetic analysis confirmed the Senegalese sole parasite as an amitochondriate Archamoeba related to Endolimax nana and Iodamoeba spp., and we tentatively describe it as a new species in the genus Endolimax, Endolimax piscium. However, the genetic distance with E. nana is quite large, with only 60% pairwise identity between both SSU rDNA genotypes. Although the overall topology of the Archamoebae cladograms containing E. piscium was consistent, the support for the branching of Endolimax spp. relative to its closest neighbours was variable, being higher with distance or parsimony-based inference methods than with ML or Bayesian trees. The use of stringent alignment sampling masks also caused instability and reduced support for some branches, including the monophyly of Endolimax spp. in the most conservative data sets. The characterization of other Archamoebae parasitizing fish could help to clarify the status of E. piscium and to interpret the large genetic distance observed between Endolimax species.