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Flavobacterium psychrophilum is the causative agent of Bacterial Cold Water Disease (BCWD)/ Rainbow Trout Fry Syndrome (RTFS) in rainbow trout Oncorhynchus mykiss. The disease is associated with significant mortality in trout farms and thereby responsible for severe economical losses in this part of the aquaculture industry. Vaccination of the very young life cycle stages is not successful due to the immature development of the adaptive immunity in yolksac larvae and early fry. This explains the extensive usage of antibiotics for control of BCWD/RTFS. Selective breeding of RTFS resistant fish may be a solution, and identification of markers associated with natural susceptibility/resistance to the disease may elevate breeding efforts towards more robust strains. We suggest a QTL (SNP Affx-88941461) for partial disease resistance on chromosome 25 (Omy25) based on our experimental F. psychrophilum challenges of outbred fish and subsequent GWAS analyses. The favourable SNP allele was designated Q, whereas q indicated the unfavourable allele. We validated this QTL in two subsequent challenge experiments by F. psychrophilum exposure of QQ, Qq and qq trout. In the first trial we produced trout carrying at least one favourable allele (QQ and Qq) associated with partial resistance by fertilizing trout eggs from non-selected (outbred) females with sperm from homozygous QQ males. In the second trial we also produced homozygous offspring by fertilizing trout eggs from QQ females with sperm from QQ males. The resistance profiles of these groups were then determined in the laboratory by triplicate or duplicate challenge experiments (common garden water bath exposure to F. psychrophilum), which showed a significantly higher survival in trout carrying the favourable allele. Field observations supplemented the laboratory studies. Under farm conditions QQ and Qq trout showed a higher survival rate compared to qq trout. In the present study chromosome 25 (Omy25) QTL was found associated with a partial resistance to F. psychrophilum. However, we recommend to include additional SNPs in future breeding studies because several QTL are associated with resistance.