Abstract: Targeting Induced Local Lesions In Genomes (TILLING) is a newly developed high-throughput reverse genetic method used for detection of point mutations. We used TILLING technology to screen EMS treated tobacco mutant population. The concentration of forward and reverse fluorescent labeled primers was determined: the 10 μL PCR mix consisted of 0.32 ~ 0.64 μL (1 μM) fluorescence-labeled forward primer and 1.12 ~ 1.6 μL (1 μM) fluorescence-labeled reverse primer. The 10 μL PCR product was used as a substrate for the CEL I digestion. The concentration of CEL I enzyme and 10× buffer optimized to cut the heteroduplex was 0.6 μL and 1.5 μL respectively. This optimized and established TILLING system was applied to screen the NtPhyB gene in 1000 (0.6% EMS mutagenized) M2 mutant population. The screening procedure provided a total of 13 mutants. The gene mutation frequency of approximately 1/94 kb was obtained. TILLING screening system established in our study is faster and efficient for mutant genes of any known sequence and can play a vital role in tobacco functional genomics research in future.