The crossability of common wheat with alien species, e.g., rye, wild and cultivated barley, is known to be controlled by the Kr gene family. The reproduction barrier caused by Kr genes decreases hybrid seed set; however, the molecular mechanism is still unclear. We attempted to localize the QTLs controlling the crossability of wheat in wheat-rye crosses by using molecular markers on wheat chromosome 5B on which the most effective Kr gene is known to be located. QTL mapping was carried out using the F₇ population derived from a cross between Chinese Spring (high crossability) and a chromosome substitution line of Chinese Spring which has its chromosome 5B of Cheyenne (low crossability), and pollinated with rye cv. Petkus. In this population, a major QTL region controlling crossability with rye was detected on the locus closely linked to a SSR marker, Xgwm443, on the short arm of chromosome 5B which was supposed to be Skr locus.