Jaguar Panthera onca populations have declined severely in Mexico because of habitat loss and poaching of the species and its natural prey. One of the most important, but poorly known, populations of the jaguar remaining in Mexico resides in the Greater Lacandona Ecosystem in Chiapas. Our objective was to determine the density of jaguars in southern Montes Azules Biosphere Reserve and to estimate population size inside the Natural Protected Areas of this Ecosystem. Jaguar densities were estimated during the dry and rainy seasons of 2007 and the dry season of 2008 using camera-trapping combined with closed capture-recapture models. The lowest density estimate was recorded during the 2007 dry season (1.7 ± SE 0.7 per 100 km2) and the highest during the 2008 rainy season (4.6 ± SE 1.6 per 100 km2). Estimating the extent of potential jaguar habitat in the Natural Protected Areas and extrapolating density estimates to these reserves indicates that they could support 62–168 jaguars. This result highlights the potential importance of this Ecosystem for the conservation of the jaguar in the Mayan Forest and Mexico. The implementation of measures to secure the long-term conservation of this population and jaguar population connectivity in the Mayan Forest is urgently required.