The aim of this study was to investigate the effect of allele variation of Ppd-B1 and Vrn-A1 on flowering time of two-line hybrid wheat parents. Five pairs of KASP functional markers were used to detect the composition and distribution of alleles in 453 two-line hybrid wheat parents; the time of sowing to flowering and growing degree days was evaluated the effect of genotypes and sowing date on the flowering time of two-line hybrid wheat parents. The results showed that the photoperiodic gene Ppd-B1 was mainly non truncated, while the vernalization genes Vrn-A1 were mainly winter, which including 36 combinations of genotypes. Among which, the groups Ⅰ, Ⅱ, Ⅷ and XI existed in both male sterile lines and restorer lines, and which were accounting for 93.62% and 52.93% in the male sterile lines and restorer lines, respectively. The sowing date had a significant effect on the time of sowing to flowering and growing degree days, while allele variation did not. The growing degree days were more stable than the time of sowing to flowering between different grown seasons and genotypes. We found that the growing degree days was stable in the two grown seasons of 2018-2019 and 2019-2020, which were 52.27 °C d and 54.02 °C d、74.44 °C d and 75.46 °C d in the combination of group Ⅲ×Ⅰ, Ⅷ×Ⅰ, in a very significant level. The result provides a scientific basis for selecting the wheat germplasm with stable flowering time by assistant of KASP molecular markers, and synchronization of flowering time by sowing date. The study also lay a scientific foundation for obtaining combinations of the two-line hybrid wheat parents with stable seed production yield.