Abstract Background Flowering time greatly influences the adaptation of wheat cultivars to diverse environmental conditions and is mainly controlled by vernalization and photoperiod genes. In wheat cultivars from the Yellow and Huai Valleys, which represent 60%-70% of the total wheat production in China, the large-scale genotyping of wheat germplasms has not yet been performed in terms of vernalization and photoperiod response alleles, limiting the use of Chinese wheat germplasms to a certain extent. Results In this study, 173 winter wheat cultivars and 51 spring wheat cultivars from China were used to identify allelic variations of vernalization and photoperiod genes as well as copy number variations of Ppd-B1 and Vrn-A1. Two new co-dominant markers were developed in order to more precisely examine Vrn-A1b, Vrn-B1a, and Vrn-B1b. Two novel alleles at the Vrn-B3 locus were discovered and were designated Vrn-B3b and Vrn-B3c. Vrn-B3b had an 890-bp insertion in the promoter region of the recessive vrn-B3 allele, and Vrn-B3c allele had 2 deletions (a 20-bp deletion and a 4-bp deletion) in the promoter region of the dominant Vrn-B3a allele. Cultivar Hemai 26 lacked the Vrn-A1 gene. RT-PCR indicated that the 890-bp insertion in the Vrn-B3b allele significantly reduced the transcription of the Vrn-B3 gene. Cultivars Chadianhong with the Vrn-B3b allele and Hemai 26 with a Vrn-A1-null allele possessed relatively later heading and flowering times compared to those of Yanzhan 4110, which harbored recessive vrn-B3 and vrn-A1 alleles. Through identification of photoperiod genes, 2 new polymorphism combinations were found in 6 winter wheat cultivars and were designated Hapl-VII and Hapl-VIII, respectively. Distribution of the vernalization and photoperiod genes indicated that all recessive alleles at the 4 vernalization response loci, truncated “Chinese Spring” Ppd-B1 allele at Ppd-B1 locus and Hapl-I at the Ppd-D1 locus were predominant in Chinese winter wheat cultivars. Conclusion This study illustrated the distribution of vernalization and photoperiod genes and identified 2 new Vrn-B3 alleles, 1 Vrn-A1-null allele, and two new Ppd-D1 polymorphism combinations, using developed functional markers. Results of this study have the potential to provide useful information for screening relatively superior wheat cultivars for better adaptability and maturity.