Stingless bees are important pollinators of plants, and also producers of honey. Species within the African stingless bee genus Hypotrigona are difficult to differentiate due to morphological similarities. Chemical profiles of whole head extracts from workers of three Hypotrigona species: H. gribodoi, H. araujoi and H. ruspolii were studied by gas chromatography–mass spectrometry. A total of 50 components belonging to six chemical classes: hydrocarbons, aldehydes, alcohols, terpenoids, steroids and fatty acids were identified. Twenty-nine compounds were found in H. araujoi, 26 in H. gribodoi and 33 in H. ruspolii head extracts. Hydrocarbons, alcohols and fatty acids were the major classes, whilst steroids and terpenoids were minor. Aldehydes were found only in H. ruspolii while terpenoids were only present in extracts of H. gribodoi and H. araujoi. Eight chemical compounds were specific to H. araujoi, six to H. gribodoi and nine to H. ruspolii, showing both qualitative and quantitative differences. Workers were successfully grouped into their respective species using their chemical profiles. This study shows that head extracts can be used as a reliable taxonomic tool for identifying and differentiating Hypotrigona species.