α-Amylases from Bacillus licheniformis (BLA) and Bacillus amyloliquefaciens (BAA) are both important industrial enzymes with high similarity in structure but significant differences in thermostability. The mechanisms underlying this discrepancy are still poorly understood. Here, we investigated the role of two amino acids' insertion on the thermostability of these two group amylases. A newly obtained thermophilic amylase AMY121 was found much closer to BLA in both primary structure and enzymological properties. Two amino acids' insertion widespread among BAA group α-amylases was identified as one of the key factors leading to the thermostability differences, since thermostability of insertion mutants (AMY121-EG and AMY121-AA) from AMY121 significantly decreased, while that of deletion mutant from BAA increased. Moreover, we proposed that conformational disturbance caused by insertion mutation might weaken the calcium-binding affinity and consequently decrease the enzyme thermostability.