Fifteen unique cDNA clones that encode trypsin and chymotrypsin-like proteins have been cloned from the gut of the Hessian fly [Mayetiola destructor (Say)] through a transcriptomic analysis. The clones encode proteins with predicted molecular weights ranged from 25.73 to 28.58, and pIs from 5.44 to 8.66. The deduced amino acid sequences of the fifteen proteins showed several structural features typical of serine proteases including: (i) the amino acid residues considered to determine trypsin or chymotrypsin substrate specificities; (ii) conserved histidine and serine catalytic sites; (iii) the catalytic triad; and (iv) six cysteine residues, all located at conserved positions. All these features suggested that these cDNAs encode active trypsins or chymotrypsins. The trypsin and chymotrypsin-like proteins can be sorted into five groups according to sequence conservation. Members from the same group share over 95% sequence identity while proteins between different groups are very different with only key residues essential for functions are conserved. Northern blot analysis revealed distinct expression profiles of these genes in different developmental stages of this insect.