β-Glycosidase activity was determined in midgut homogenates and in electrophoretically-resolved midgut samples from the following insects: Orthoptera, Abracris flavolineata; Coleoptera, Pheropsophus aequinoctialis, Tenebrio molitor, Pyrearinus termitilluminans; Hymenoptera, Scaptotrigona bipunctata; Diptera, Rhynchosciara americana; Lepidoptera, Erinniys ello, Spodoptera frugiperda, and Diatraea saccharalis. The substrates used in the assays included disaccharides (cellobiose and lactose), synthetic aryl β-glucosides (p-nitrophenyl β-glucoside and p-nitrophenyl β-galactoside), and plant glycosides (salicin and amygdalin). Orthopterans, coleopterans, and hymenopterans have in their midgut β-galactosidases (active only on β-galactosides) and class 2 (active only on disaccharides) and class 3 (active only on synthetic and plant glycosides) β-glucosidases. Class 1 (active on disaccharides and on synthetic and plant glycosides) β-glucosidases, at least in orthopterans, have putatively different sites for each activity. Dipterans have β-galactosidases and a single class 1 β-glucosidase. Lepidopterans have no β-galactosidases and may have a single class 1 β-glucosidase or class 1 and class 3 β-glucosidases. There seems to be an evolutionary trend from multiple enzymes with different substrate specificities to a single enzyme able to hydrolyse all the β-glycosides in the same site.