Endolimax is a genus of intestinal amoebae which stands among the least known human protists. Previous studies on amoebic systemic granulomatosis of a marine fish (Solea senegalensis) resulted in the unexpected characterization of a new organism which was related to Endolimax and named E. piscium. The existence of multiple reports of systemic granulomatosis caused presumptively by unidentified amoebae in goldfish lead us to investigate the organism involved in goldfish disease. Analysed goldfish presented small whitish nodules in the kidney, which correspond to chronic granulomatous inflammatory reactions with a ring-layer of amoebae in the periphery. Amoebae were amitochondriate and were located in a parasitophorous vacuole within macrophages, as previous studies on this condition in goldfish and other freshwater fish pointed out. SSU rDNA characterization confirmed a new Endolimax lineage which appears closely related to E. piscium, but the molecular evidence, distinct pathological features and lack of ecological overlapping between the hosts support their assignment to a new species, E. carassius. The results support the existence of a considerable unexplored diversity of Endolimax spp. among fish, and their proper characterization can contribute to an understanding of Archamoebae evolution and pathogenic potential.