The F3 h gene encoding flavanone 3-hydroxylase, one of the key enzymes of the flavonoid biosynthesis pathway, is involved in plant defense response, however, it has not yet been genetically mapped in such important crop species as wheat, barley and rye. In the current study, the F3 h genes were for the first time genetically mapped in these species, using microsatellite and RFLP markers. The three wheat F3 h homoeologous copies F3 h-A1, F3 h-B1 and F3 h-D1, and rye F3 h-R1 were mapped close to the microsatellite loci Xgwm0877 and Xgwm1067 on chromosomes 2AL, 2BL, 2DL, and 2RL, respectively. Wheat F3 h-G1 and barley F3 h-H1 were also mapped at the homoeologous F3 h-1 position on chromosomes 2GL and 2HL, respectively. The non-homoeologous F3 h gene (F3 h-B2) was mapped on wheat chromosome 2BL about 40 cM distal to the F3 h-1 map position. The results obtained in the current study are important for further studies aimed on manipulation with F3 h expression (and, hence, plant defense) in wheat, barley and rye.