CEL I-based TILLING (Targeting Induced Local Lesions In Genomes) platform is very useful for high throughput identification of point mutations within targeted genes and improvement of mutation-detected efficiency in wheat. The space-mutated Xinmai 18 (Triticum aestivum L.) SP2 population was used with one SNP of Waxy gene in the population as a positive control. The TILLING protocol for wheat was optimized through improving the method of genomic DNA extraction, the concentration of dNTP, Mg2 ,primers and CEL I buffer as well as experimental environment air humidity. It was found that through raising the grinding frequence to 30/s and prolonging the reaction time of KAc to 20min in genomic DNA extraction, the quality and purification of DNA were the best. The concentration of both dNTP and Mg2 did not have any influence on PCR products within the set ranges, while primer played an important role in PCR products and the best concentration was 0.4 umol L-1. Optimum amount of CEL I enzyme was 0.1U in a 20 祃 of digestion reaction system and ultrapure water substituting for CEL I buffer was the best reaction condition. A CEL I-based TILLING technique for wheat was established and optimized.