Flavobacterium psychrophilum mainly infects salmonids and can cause bacterial cold water disease (BCWD), which usually occurs under low temperature conditions and has a high mortality rate, seriously limiting the healthy development of the salmonid aquaculture industry in China. In this experiment, two pathogenic bacterial strains (CH06 and CH07) were isolated from the muscle lesions of diseased rainbow trout (Oncorhynchus mykiss), and their biochemical characteristics, molecular characteristics, genotypes, serotypes, drug resistance and pathogenicity were analyzed. The results showed that CH06 and CH07 isolates were highly pathogenic to juvenile rainbow trout with the LD₅₀ values of 7.1 × 10^₅ and 1.1 × 10^₅ CFU / mL, respectively. The dosage of the challenge was inversely proportional to the time when the clinical signs appeared. CH06 and CH07 isolates had an egg-like appearance on TYES agar, produced flexirubin-like pigments, and were positive for oxidase and catalase. It can hydrolyze gelatin and casein, cannot hydrolyze starch or use fructose, galactose and esculin. The results of 16S rRNA gene alignment suggested that the homology of CH06 and CH07 isolates with the Flavobacterium psychrophilum reference strain NBRC15942 strain were 99.35% and 99.42%, respectively. Based on the biochemical and molecular characteristics results, CH06 and CH07 isolates were identified as Flavobacterium psychrophilum. The multiplex PCR method was used to identify the serotypes of CH06 and CH07 isolates as type 1 (Fd type). MLST analysis indicated that the genotypes of CH06 and CH07 isolates were ST-12 and ST-78, respectively. CH06 and CH07 isolates exhibited slightly different resistance profiles to 10 antibacterial drugs. They were both highly susceptible to ampicillin and trimethoprim-sulfamethoxazole, while CH06 isolate was reduced susceptible to enrofloxacin and florfenicol and CH07 isolate was moderately susceptible to enrofloxacin and florfenicol. Histopathological changes showed that the hepatocytes of the diseased fish were swollen and vacuoles degenerated. Some hepatocytes were lysed and necrotic and the nucleus was dissolved. The spleen was congested and hemorrhagic with reduced amounts of the lymphocytes and increased amounts of the red blood cells and the hemosiderin. The muscle fiber gap was widened and broken, uneven bending. Parts of the muscle cell sarcoplasmic dissolution was honeycomb-like. For the first time, this experiment detected Flavobacterium psychrophilum from domestically cultured rainbow trout, and this initial study on the taxonomic status, pathogenicity and drug resistance of the pathogens were expected to provide a scientific basis for the prevention and control of bacterial cold water disease in rainbow trout.