The genetics and ecology of pumas are well documented in North America but there is a lack of studies in South America, especially in Brazil. By means of a noninvasive method, faecal DNA analysis, we estimated puma abundance in two protected areas embedded in a human-disturbed landscape in the north-east of São Paulo state, in south-east Brazil. In 8 months of mark–recapture faeces sampling, 15 individual pumas were identified using seven microsatellite loci. The estimated abundance of pumas with the Jolly–Seber open population model was 23.81 ± SE 6.22. This is the first estimate of the abundance of pumas in a human-dominated landscape in São Paulo state, the most populous, developed and industrialized state of Brazil. The absence of high-quality habitats in the north-east of the state, the absence of direct competitors and the high availability of prey in protected areas are probably contributing to the high number of pumas concentrated in a relatively small area (c. 260 km2). Our results will contribute to the long-term monitoring of this puma population and, combined with other ecological, behavioural and genetic data, will help guide conservation action to maintain a viable population of the puma in this region.