Two strains resembling the neurotropic fungus Ochroconis gallopava were isolated from hot spring river water (IFM 54738 and IFM 54739). The isolates showed optimal growth at 42 degrees C, while the maximum growth temperature was 49 degrees C, thus having temperature relationships similar to those of O. gallopava. Colonies were light olive green, with a color change to dark reddish brown after several passages, which was also observed in O. gallopava. Conidia were indistinguishable from those of O. gallopava. The antifungal susceptibility profile of the isolates was also similar to that of O. gallopava, except for a lower susceptibility to micafungin. The two isolates had 100% homologous rRNA genes including the internal transcribed spacer (ITS) region and the D1/D2 region of the large subunit. The gene fragments, as O. gallopava, could be amplified with species-specific rDNA primers, and loop-mediated isothermal amplification designed for O. gallopava yielded positive results in the two isolates. However, homologies with O. gallopava in ITS and D1/D2 regions were 79.2 and 95.9%, respectively, widely exceeding generally accepted species boundaries. These differences were corroborated in virulence tested in experimental infection. The two isolates did not kill a mouse even until 28 days. However, mortalities of four O. gallopava strains ranged from 40 to 100%. The new isolates mainly affected the kidneys; whereas O. gallopava had a strong preference for the brain. We therefore propose a new species, Ochroconis calidifluminalis, for the two isolates.