Jaguar population genetics has so far not been investigated on a broad scale in the Amazon rainforest, which constitutes the largest remaining block of continuous habitat for the species. Given its size, it serves not only as a stronghold but also as a reference for jaguar conservation genetics, against which fragmented landscapes can be compared. We assessed genetic diversity and population structure of Amazonian jaguars using 11 microsatellite loci and performed comparative analyses incorporating available data from two other South American biomes (Pantanal and Atlantic Forest) in which the species has faced different amounts of habitat loss and fragmentation. Using the largest genetic data set assembled to date for jaguars (n = .190), we observed that all diversity indices were consistently higher for the Amazonian population, with no genetic subdivision detected in that region, indicating large-scale connectivity across >3000 km. In contrast, we corroborate the inference of anthropic-driven genetic structure and bottlenecks for two Atlantic Forest populations. Our results indicate that the Amazon is a critically important stronghold for jaguars, comprising a highly diverse, panmictic population that allows a glimpse into the patterns of genetic connectivity that characterized this species prior to human intervention. In contrast, the Atlantic Forest populations jointly still retain considerable levels of genetic diversity, but this is currently partitioned among isolated fragments that are increasingly subjected to heavy anthropic disturbance. These results have important implications for jaguar conservation planning, highlighting the critical condition of Atlantic Forest populations and providing a genetic baseline to which they can be compared.