Abstract: Alpha-amylase inhibitors from wheat are found noble sources to develop insect resistant transgenic plants. Thus far wheat alpha – amylase inhibitor genes have been cloned, sequenced and characterized by various researchers through c-DNA library. A subunit of tetrameric alpha amylase CM3 gene was isolated and characterized directly from genomes of different Indian wheat varieties using PCR approach. We have screened different Indian wheat varieties for alpha amylase inhibitor protein(s) and isolated the gene from selected variety to develop transgenic plant having gene of better expressivity and enhanced activity. The alpha amylase inhibitor gene encoding CM3 protein, from T. aestivum and T. durum has cloned and sequenced, the gene sequences from the both species were found same except at one position which is out of coding region. Further, characterization reveals that the isolated gene is intronless and conserved throughout the different varities of T. aestivum and T. durum. It may be possible that there is no ecological or geographical effect on the gene. This indicates probability of important biological role for this protein in wheat.