The maned wolf is the largest South American canid, and in Brazil, its distribution is closely associated with the Cerrado, one of the most threatened biomes in the world. Although the Cerrado have few, small, and often isolated protected areas, these fragments are crucial for the persistence of wide-ranging mammals, providing protection and resources, and facilitating their dispersal movements, especially in areas of intense human activity. Here, we estimated the minimum population size and genetic diversity of maned wolves inhabiting one of the last refuges of Cerrado vegetation in São Paulo State, the most developed, industrialized, and populous state of Brazil. Through fecal DNA analysis of five microsatellite loci, during a 13-month sampling period, we identified 13 maned wolves and observed a genetic diversity similar to other studies conducted in larger and better preserved areas of the central region of the country. However, unlike those studies, we found evidence of inbreeding, deviations from Hardy-Weinberg expectations, and a larger number of animals in relation to the sampled area, thus providing evidence that the population may be threatened.