Beetles of the Passalidae family live and feed on decaying wood and their guts are richly colonized by yeasts. The goal of this research was to prospect xylolytic yeasts with potential for the production of second-generation bioethanol. Therefore, 83 specimens of beetles belonging to the Passalidae and the Scarabaeidae families were collected in the Amazonian rainforest in Itacoatiara -AM, Brazil. Morphological differences of the beetles were identified and 25 chosen specimens were dissected. Yeasts from galleries inhabited by beetles and from insect guts were isolated. Isolates were previously selected through tolerance tests for temperature, ethanol and xylose assimilation capacity. Those isolates were then submitted to a panel of conditions related to ethanol production. The ethanol production reached 24.70 g.L -1 and the xylitol production reached 21.66 g.L -1. One of the isolates with a promising profile was identified as Spathaspora roraimanensis and six as Spathaspora passalidarum. Three isolates showed to be more promising and, curiously, all came from the gut of the species Popilius marginatus (Percheron, 1835). In plate testing, however, the isolates obtained from galleries showed a greater capacity to assimilate xylose. As reported in this field of study, no isolate tolerated all conditions tested. Wild isolates with this profile may be used for testing larger-scale ethanol production, genetic engineering, or evolutionary techniques.