Midgut expressed alkaline serine proteases of Lepidoptera function in conversion of Bacillus thuringiensis (Bt) protoxin to active toxin, and reduced level of transcript T23 is associated with Ostrinia nubilalis resistance to Dipel® Bt formulations. Three groups of trypsin- (OnT25, OnT23, and OnT3) and two chymotrypsin-like (OnC1 and OnC2) cDNAs were isolated from O. nubilalis midgut tissue. Intraspecific groupings are based on cDNA similarity and peptide phylogeny. Derived serine proteases showed a catalytic triad (His, Asp, and Ser; except transcript OnT23a), three substrate specificity-determining residues, and three paired disulphide bonds. RT-PCR indicated all transcripts are expressed in the midgut. Mendelian-inherited genomic markers for loci OnT23, OnT3 and OnC1 will be useful for association of alleles with bioassayed Bt toxin resistance phenotypes.