Esterases form a large, diverse group of enzymes with wide, overlapping substrate specificities and patterns of inhibition. These enzymes occur in a large variety of isoforms encoded by distinct gene loci with a high genetic variability and temporal differences in expression that make them appropriate for studying population structure. In this work, we investigated the substrate specificity and pattern of esterase expression in body parts of the stingless bees Tetragonisca angustula and Tetragona clavipes. Fourteen hives of T. angustula were collected in Cianorte and T. clavipes were collected from three colonies in Maringá, two cities in the southern Brazilian state of Paraná. The esterase electrophoretic patterns were determined using polyacrylamide gels. Seven bands of esterase activity were detected in T. clavipes (EST-1 to EST-7) and two bands in T. angustula (EST-1 and EST-2). There was variation in the tissue esterase activity of T. clavipes, with EST-6 occurring in the abdomen of workers and EST-7 occurring in cephalic/thoracic extracts. The differences in the number of esterase bands and substrate specificity were attributed to the number of esterase loci involved in each species, and/or variation in substrates specificities. The variation seen here should be useful for determining the role of esterases in intermediate metabolism in the Trigonini, as well as to use esterases as a genetic marker for this stingless bee.