Wheat stripe rust is one of the most damaging diseases of wheat worldwide. The wheat-Leymus mollis introgression line M8664-3 exhibits all-stage resistance to Chinese stripe rust races. Genetic analysis of stripe rust resistance was performed by crossing M8664-3 with the susceptible line Mingxian169. Analysis of the disease resistance of F2 and F2:3 populations revealed that its resistance to Chinese stripe rust race 33 (CYR33) is controlled by a single dominant gene, temporarily designated as YrM8664-3. Genetic and physical mapping showed that YrM8664-3 is located in bin 4AL13-0.59-0.66 close to 4AL12-0.43-0.59 on chromosome 4AL and is flanked by single-nucleotide polymorphism markers AX111655681 and AX109496237 with genetic distances of 5.3 and 2.3 centimorgans, respectively. Resistance spectrum and position analyses indicated that YrM8664-3 may be a novel gene. Molecular detection using the markers linked to YrM8664-3 with wheat varieties commonly cultivated and wheat-L. mollis-derived lines showed that YrM8664-3 is also present in other wheat-L. mollis introgression lines but absent in commercial common wheat cultivars. Thus, YrM8664-3 is a potentially valuable source of stripe rust resistance for breeding.