During a large-scale bacterial culturing effort of biofilms in the vicinity of a rainbow trout aquaculture facility in Idaho, USA, 10 isolates were identified as having pathogen-inhibiting activity and were characterized further. These isolates were shown to be Gram-negative, rod-shaped bacteria belonging to the genus Pseudomonas . Whole-genome comparisons and multi-locus sequence analysis using four housekeeping genes (16S rRNA, gyrA, rpoB and rpoD) showed that these 10 isolates clustered into four distinct species groups. These comparisons also indicated that these isolates were below the established species cutoffs for the genus Pseudomonas . Further phenotypic characterization using API 20NE, API ZYM and Biolog GENIII assays and chemotaxonomic analysis of cellular fatty acids were carried out. Based on the genomic, physiological and chemotaxonomic properties of these isolates, we concluded that these strains composed four novel species of the genus Pseudomonas . The proposed names are as follows: Pseudomonas aphyarum sp. nov. consisting of strains ID233, ID386T and ID387 with ID386T (=DSM 114641T=ATCC TSD-305T) as the type strain; Pseudomonas rubra sp. nov. consisting of strains ID291T, ID609 and ID1025 with ID291T (=DSM 114640T=ATCC TSD-303T) as the type strain; Pseudomonas idahonensis sp. nov. consisting of strains ID357T and ID1048 with ID357T (=DSM 114609T=ATCC TSD-304T) as the type strain; and Pseudomonas fontis sp. nov. consisting of strains ID656T and ID681 with ID656T (=DSM 114610T=ATCC TSD-306T) as the type strain.