Salmonid diseases caused by infections of Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease, remain difficult to manage as novel, pathogenic strains continue to emerge in aquaculture settings globally. To date, much of the research regarding treatment options and vaccine development has focused on rainbow trout (Oncorhynchus mykiss), but other inland‐reared salmonids are also impacted by this Gram‐negative bacterium. As such, Atlantic salmon (Salmo salar) and brook trout (Salvelinus fontinalis) were injection‐challenged with a variety of previously reported F. psychrophilum strains isolated from disease diagnostic cases in salmonids, as well as a standard and well‐studied F. psychrophilum strain (CSF 259–93) known to be virulent in rainbow trout. In three separate virulence assessments (Trials A, B and C), strains US063 (isolated from lake trout; Salvelinus namaycush) and US149 (isolated from Atlantic salmon) caused a significantly higher cumulative per cent mortality (CPM) relative to other strains in Atlantic salmon (p <.001 for all trials), with US149 causing significantly greater mortality than US063 in Trials A (CPM 97% vs. 65%, p =.008) and B (CPM 96% ± 2.3% vs. 81.33% ± 4.8%, p =.014). Trial C used a lower dose (1.86 × 108 CFU/mL) for US149, resulting in a lower mortality (78.67% ± 9.33%) relative to Trials A and B. CSF259‐93 did not cause significant mortality in any trials. In brook trout, the strain 03–179 (originally isolated from steelhead trout; Oncorhynchus mykiss) was significantly more virulent than any other (CPM 100% ± 0%, p <.001), followed by US063 (73% ± 3.8%) and US149 (40% ± 6.1%,) respectively. Again, CSF259‐93 did not cause significant mortality relative to a mock challenge treatment. Results provide information about the applicability of strain selection in F. psychrophilum virulence testing in Atlantic salmon and brook trout, demonstrating the high virulence of US063 and US149 for these salmonid species. This information is applicable for the development of therapeutics and vaccines against F. psychrophilum infections and demonstrates the reproducibility of the experimental challenge model.