l-Amino acid α-ligase (Lal), catalyzing the formation of α-dipeptides from unprotected l- amino acids in an ATP-dependent manner, is used in cost-effective fermentative production of dipeptides. We searched for novel Lals by in silico screening using Hidden Markov Model- based profile analysis, and identified five novel Lals that showed low similarity and different substrate specificity from known Lals.