Reproductive stage frost poses a major constraint for wheat production in countries such as Australia. However, little progress has been made in identifying key genes to overcome the constraint. In the present study, a severe frost event hit two large-scale field trials consisting of six doubled haploid (DH) wheat populations in reproductive stage (young microspore stage) in Western Australia, leading to the identification of 30 robust frost QTL. Results showed that frost damage is associated with dominant alleles of VrnA1a, VrnD1a, Rht-B1b, Rht-D1b, and the copy number of Ppd-B1. No frost QTL was detected on chromosomes 5B and 1B, although anthesis QTL and anthesis related genes of VrnB1a and TaFT3_1B were co-detected, indicating that these early-flowering phenotype inducing genes are compatible with frost tolerance and thus can be utilised in breeding. Our results also indicate that wild-type or recessive alleles Rht-B1a (rht1) and Rht-D1a (rht2) can be used when breeding for frost-tolerant varieties without delaying flowering time.