In this contribution, the first observations of euglossine bee species from the Bolivian Prepuna are reported. Euglossa melanotricha Moure, Eufriesea mariana (Mocsáry) and Exaerete dentata (L.) were observed at an elevation of 2640 m in a garden located in San Joaquin, Salancachi (Chuquisaca department, annual precipitation 400–500 mm). This is the highest known record for a population of Euglossa and Eufriesea to date. All of the euglossine bees were observed exclusively on introduced plants. The distributional patterns, biological associations and ecological significance of the observed euglossine bees are discussed.