In our previous study, CD-3, a novel CS-Dasypyrum villosum 3V (3D) substitution line with high resistance to wheat stripe rust, had been developed, and the stripe rust resistance had been mapped on the 3V chromosome. To explored the transmission character of the 3V chromosome and provide the theoretical basis for the utilization of stripe rust resistance from 3V in wheat breeding, CD-3 was directly and reciprocally crossed with landrace Kaixian Luohan Mai wheat (KL) and commercial variety Chuan Mai 42 wheat (CM42), respectively, in this study. The F1 hybrids were then selfed to obtain F2 plants for estimation of the transmission characters of the 3V chromosome using fluorescent in situ hybridization (FISH). At last, the stripe rust resistance of the F2 plants was investigated. The results showed: (1) The transmission rates of 3V in the 4 crosses (CD-3/KL, KL/CD-3, CD-3/CM42 and CM42/CD-3) ranged from 20.45% to 34.94%, which were significantly lower than the theoretical value (75%), and there was no significant difference among the 4 crosses, indicating that the genetic background had no influence on the transmission of 3V in this study; (2) The chromosomal structural aberration rate of 3V in CD-3/CM42 was significantly higher than that in CD-3/KL and CM42/CD-3, suggesting that the genetic background had influence on the chromosomal structural aberration rate of 3V; (3) The plants with two 3V chromosomes showed high resistance, and the plants carrying one 3V chromosome were susceptible or moderately resistant to stripe rust, which was consistent with the infection type of their parents, CM42 and KL. The result showed that the resistant gene(s) of stripe rust located on the 3V chromosome could express in the genetic background of the landrace KL and the commercial variety CM42. This research provided theoretical foundation for further exploration of the resistant gene(s) of stripe rust originating from the 3V chromosome of D. villosum, as well as valuable genetic materials for wheat improvement.