Flavobacterium psychrophilum is the pathogen of bacterial cold water disease (BCWD). Its prevalence and occurrence seriously restrict the healthy development of the salmon and trout industry. This study explored the tissue and dynamic distribution of F. psychrophilum in fish after intramuscular injection of infected rainbow trout (Oncorhynchus mykiss) to provide theoretical support for the prevention and control of BCWD. Experimental rainbow trout was infected intramuscularly with 1.0×10⁸ CFU/mL concentration of F. psychrophilum CH06 strain. The clinical symptoms of rainbow trout were observed at 12 h, 24 h and 96 h after infection, and real-time PCR was performed using the Taqman probe method to detect the dynamic distribution of pathogens in different tissues of fish. The clinical symptoms of the CH06 strain infection fish are blackening of body surface, slow or immobile swimming, loss of appetite, muscle ulceration at the injection site of the tail handle, pale gills, splenomegaly, and ascites. Histopathological observations showed that the muscle fibers were broken and dissolved in experimental fish. The splenic sinuses were dilated and filled with erythrocytes. There was an increase in haemosiderin in the kidney, with massive vacuolar degeneration, necrosis of the renal tubular epithelium and infiltration of many inflammatory cells in the renal interstitium. qPCR results showed that after 12 h of intramuscular injection, F. psychrophilum could be detected in the spleen, liver, kidney, intestine, gills, muscle at the injection site, brain and caudal fin. The highest load of muscle pathogen at the injection site was (5.85±2.11) ×10⁵ copy/μL. After 24 h of infection, the pathogen load in the spleen and brain increased most compared to 12 h of infection. The load of pathogens in liver, kidney, intestine, gills, muscle at the injection site and caudal fin was consistent with the level at 12 h of infection. The load of muscle pathogen at the injection site was (6.48±2.07) ×10⁵ copy/μL, significantly higher than spleen, liver, kidney, intestine, gills, brain and caudal fin (P＜0.05). After 96 h of infection, the pathogen load in the spleen was significantly higher than liver, kidney, intestine, gills, muscle at the injection site, brain and caudal fin (P＜0.05), reaching (1.15±0.58)×10⁷ copy/μL; the pathogen load in the liver, kidney, and spleen increased most than at 24 h of infection. The pathogenic bacterial load in all tested tissues showed an upward trend with time. In addition, the average pathogen load of the muscle at the injection site was highest at the three time points, followed by the spleen, kidneys, and gills. After artificially infecting rainbow trout with F. psychrophilum, the muscle at the injection site and spleen are important proliferation sites for bacteria. In summary, the pathogen entered various tissues along with blood circulation, and exhibited strong tropism to the muscle at the injection site, spleen, kidney and gills tissue, but exhibited week tropism to liver, caudal fin, intestine and brain tissue. The infection time and pathological changes were positively correlated with the pathogen load in tissues.