Regeneration of cultured tissue is a prerequisite of Agrobacterium- and biolistic-mediated plant transformation. In this study, an efficient protocol for improving wheat (Triticum aestivum L.) immature embryo regeneration was developed. Based on the statistical analysis of embryogenic callus induction efficiency, green spot differentiation efficiency, and plant regeneration efficiency from five wheat accessions, improved culture conditions were found to be more effective for embryogenic callus production than the traditional conditions. Using semi-quantitative reverse transcription polymerase chain reaction, a candidate gene, designated as TaCAT1, which encodes a catalase was identified to have a significant correlation with high-regeneration trait of wheat immature embryos. Three amino acid substitutions were found in TaCAT1 protein between high- and low-regeneration wheat accessions. Hydrogen peroxide content in the cultured calli increased from day 5 to 15, and then decreased sharply on day 20, followed by a second peak on day 25 during regeneration stage. Furthermore, a 3,500-bp 5' flanking region upstream of the first codon ATG of TaCAT1 was isolated using inverse polymerase chain reaction. In silico, analysis revealed that the TaCAT1 promoter contained two regulatory motifs associated with responses to auxin.