Sex pheromone production by insects aims to attract the opposite sex for mating. However, not only sexually attractive virgin Tetragonisca angustula queens release these compounds, but also mated queens. Herein, the putative sources of T. angustula queen pheromones were studied by the gas chromatography coupled to mass spectrometry (GC-MS) analyses of volatile compounds and abdominal gland, cuticular and cephalic extracts of sexually attractive virgin and physogastric queens. Isopropyl and hexyl hexanoates released by T. angustula physogastric queens were found both in their Dufour’s and tergal glands. Although both esters were present in the tergal glands of sexually attractive virgin queens, only isopropyl hexanoate was detected by the aeration method. Octadecyl octadecenoate was found to be the main constituent of the sexually attractive virgin queen’s Dufour’s glands, and this same gland contained several esters in physogastric queens. Complex mixtures of normal, branched and unsaturated hydrocarbons were found in the tergal gland and cuticular extracts. The chemical composition of the cephalic physogastric queen extract was similar to the cuticular extract, except for the presence of acids and steroids. In contrast, the virgin queen’s head was mostly composed of nerol.