Background: Connectivity among jaguar (Panthera onca) populations will ensure natural gene flow and the long-term survival of the species throughout its range. Jaguar conservation efforts have focused primarily on connecting suitable habitat in a broad-scale. Accelerated habitat reduction, human-wildlife conflict, limited funding, and the complexity of jaguar behaviour have proven challenging to maintain connectivity between populations effectively. Here, we used non-invasive genetic sampling and individual-based conservation genetic analyses to assess genetic diversity and levels of genetic connectivity between individuals in the Cockscomb Basin Wildlife Sanctuary and the Maya Forest Corridor. We used expert knowledge and scientific literature to develop models of landscape permeability based on circuit theory with fine-scale landscape features as ecosystem types, distance to human settlements and roads to predict the most probable jaguar movement across central Belize. Results: We used 12 highly polymorphic microsatellite loci to identify 50 individual jaguars. We detected high levels of genetic diversity across loci (HE = 0.61, HO = 0.55, and NA = 9.33). Using Bayesian clustering and multivariate models to assess gene flow and genetic structure, we identified one single group of jaguars (K = 1). We identified critical areas for jaguar movement that fall outside the boundaries of current protected areas in central Belize. We detected two main areas of high landscape permeability in a stretch of approximately 18 km between Sittee River Forest Reserve and Manatee Forest Reserve that may increase functional connectivity and facilitate jaguar dispersal from and to Cockscomb Basin Wildlife Sanctuary. Our analysis provides important insights on fine-scale genetic and landscape connectivity of jaguars in central Belize, an area of conservation concern. Conclusions: The results of our study demonstrate high levels of relatively recent gene flow for jaguars between two study sites in central Belize. Our landscape analysis detected corridors of expected jaguar movement between the Cockscomb Basin Wildlife Sanctuary and the Maya Forest Corridor. We highlight the importance of maintaining already established corridors and consolidating new areas that further promote jaguar movement across suitable habitat beyond the boundaries of currently protected areas. Continued conservation efforts within identified corridors will further maintain and increase genetic connectivity in central Belize.