Rice blast and bacterial blight,caused by Magnaporthe oryzae and Xanthomonas oryzae pv.oryzae,respectively,are two principal diseases worldwide.Meanwhile,these diseases are also the major limitations in rice production in China.Lijiangxintuanheigu(LTH) is a highly susceptible cultivar to rice blast,while Tetep(TTP) has broad-spectrum resistance against the pathogens.However,the molecular mechanism underlying the difference on disease resistance in LTH and TTP is largely unknown.To identify the nucleotide difference in multiple resistance genes between these two cultivars efficiently,6 known R genes were amplified from LTH and TTP,respectively.The PCR products were mixed with a 10-cycle difference in Ct value and sequenced together by the next-generation sequencing(NGS) technology.The results were validated by traditional sequencing strategy.Our results revealed that the NGS can rapidly identified nucleotide polymorphisms from multiple PCR-amplified products of different cultivars in single sequencing run,which is more economical and more efficient to find sequence difference of target genes among different cultivars.Additionally,we found that the sequence of R genes in TTP is more similar to that of original functional alleles,while the sequence of R genes in LTH has more mutations that altering amino acid.