Measurement of the electrolyte leakage rates in wheat leaves indicated that there was no significant difference in susceptibility to -5 C spring freeze stress among five bread wheat cultivars at the floret primordiumdifferentiating stage of spike development. A global transcriptional profile was created using the Affymetrix Wheat GeneChip microarray for one wheat cultivar (Yumai 34) under -5 C freeze stress. After assaying genes with significant regulation at 1 and 3 days after -5 C freeze stress, we identified 600 genes that were previously annotated as showing changes in expression of at least than two-fold at one or both of the time points. Among these genes, we further analysed 102 genes whose expression levels changed at least eight-fold after 1 or 3 days of freeze stress. These genes encoded an ice recrystallization protein, cold-related proteins, CBF transcription factors, calcium-dependent protein kinases, Na?/H? antiporters, aquaporins, and many metabolic enzymes. The results of this study were compared with those of a previous study on the sub-freeze hardening response in wheat and spring freeze stress in wheat and barley. Many genes, including those encoding WCOR413, LEA, glycine-rich RNA-binding protein, ferritin, aquaporin 2, and a pathogen-induced protein, showed similar expression levels in these studies. Spring freeze stress is a complex phenomenon involving physiological mechanisms and multiple genes that had not been previously characterised.