The purpose of this study is to expand our knowledge of the diversity of lobose amoebae (Amoebozoa) that can be isolated from the gills of farmed rainbow trout (Oncorhynchus mykiss Walbaum, 1792) affected by nodular gill disease (NGD). A new strain of an amoebozoan, Ptolemeba bulliensis Watson et al. 2014 (Tubulinea) was isolated, studied and identified from the gills of farmed trout showing signs of NGD in the northwest of Russia. The strain was identified using morphological and molecular (small subunit [SSU] ribosomal RNA gene sequence data) characters. Actin and cytochrome c oxidase (Cox1) gene sequences were obtained for the representative of Ptolemeba for the first time. A peculiar feature of the Cox1 gene is the presence of group I intron that contains a 177 amino acids long open reading frame encoding a homing endonuclease. This is the first case of a group I intron in the Cox1 gene in Amoebozoa apart from Dictyostelium. In the experimental study, the studied amoebae demonstrate a broad range of salinity tolerance. They are capable of survival and reproduction in a range of salinities between freshwater Prescott and James medium and 18‰, but could not survive or reproduce in 25‰ and above. This indicates a possibility of this species' distribution through brackish water biotopes and shows that in case of pathogenicity for the farmed fish, the methods of treatment based on increasing salinity may be inefficient. In spite of the lack of direct evidence for pathogenicity, repeated observations of P. bulliensis isolated from the gills may suggest its association with the disease.