The polyacrylamide gel electrophoresis system (PAGE) and inhibition tests for biochemical characterization of α- and β-esterases were used to obtain a functional classification of esterases in plants and to show a differential expression of esterases as markers of pathogenesis in cassava plants (Manihot esculenta Crantz). The characterization of α- and β-esterases from leaves of M. esculenta by the PAGE system was possible using an extraction solution containing two phenol-complexing agents (PVP-40 and sodium metabisulfite), three antioxidant agents (EDTA, β-mercaptoethanol, and DTT), and one quinone reducer (ascorbic acid). Fourteen esterase isozymes were detected in young unexpanded leaves of M. esculenta cultivars. The inhibition pattern of α- and β-esterases of M. esculenta showed that Est-9 is an arylesterase, and in the unexpanded leaves of the M. esculenta plants infected with Xanthomonas axonopodis pv. manihotis, the Est-7 β-esterase showed the characteristic staining of an α/β-esterase. This differential expression of Est-7 isozyme in young unexpanded leaves of cassava plants can be used as a marker of pathogenesis after infection with X. axonopodis pv. manihotis.