Understanding the genetic structure of hexaploid wheat (Triticum aestivum L.) is important to develop useful and effective methods for genetic analyses. Particularly frost tolerance (FT) in wheat is essential for preventing yield losses. Therefore, the objectives of this study were development of genome specific primers of FT candidate genes for re-sequencing, polymorphism detection and candidate gene based association analysis. A set of 39 specific primer pairs corresponding to 19 candidate genes were developed with a success rate of >50%. The genes corresponding to C-REPEAT BINDING FACTORS (CBFs), i.e. CBF-A3, CBF-A5, CBF-A10, CBF-A13, CBF-A14, CBF-A15, CBF-A18, as well as the VERNALISATION RESPONSE GENES VRN-A1, VRN-B3, and the PHOTOPERIOD RESPONSE GENES PPD-B1 and PPD-D1 revealed associations to FT in 235 wheat cultivars. Six of these genes exhibit amino acid (AA) substitution polymorphisms in important protein domains. The effect on FT based on AA substitution in VRN-A1 is also described in related literature, but the AA substitutions in CBFA3, CBF-A5, VRN-B3, PPD-B1 and PPD-D1 were yet unknown.