Although it has been hypothesized that men and women vary in the way they value ecosystem services, research on ecosystem services rarely incorporates a gender dimension. We conducted research with nine indigenous communities in the Colombian Amazon to understand which ecosystem services men and women perceive as most important for their wellbeing and to rank them according to locally-defined criteria of importance. Participants identified a total of 26 ecosystem services and 20 different ranking criteria. Ecosystem services such as land for agricultural fields (a supporting service), and provision of fish and medicinal plants were equally important for both men and women. Wild fruits and resources to make handicrafts were more frequently mentioned by women, whereas timber, materials for making tools and coca leaves were more frequently mentioned by men. There were also differences in the criteria used to value ecosystem services, with 11 criteria mentioned by both men and women, five mentioned exclusively by women and another four only by men. Our results suggest that taking gender differences into account in ecosystem services assessments may result in the prioritization of different services in conservation and sustainable development programs, and may lead to different outcomes for ecosystem service provision and local livelihoods.