Many carnivore species, particularly felids, are sensitive to land use changes and may disappear from landscapes with reduced natural habitat and increased fragmentation. The jaguar (Panthera onca) is highly affected by these factors and is particularly endangered in the Atlantic Forest (AF) of South America, one of the most threatened biomes in the world. We used noninvasive fecal sampling and microsatellite markers to investigate the genetic diversity of jaguars in one of the last remnant populations of this species in the entire coastal AF. This section of the biome is highly fragmented. We observed low levels of genetic diversity (H O = 0.621, H E = 0.532, AR = 3.195), and estimated a small effective size for the population (N e = 7.9 individuals). We performed comparative analyses incorporating data from previously surveyed populations located farther inland (interior AF), revealing that the coastal population studied shows significant genetic differentiation. Our results support a scenario of anthropogenic, drift-induced differentiation among jaguar populations in small AF fragments, affecting both the interior and coastal components of this biome. Despite its low diversity, we detected putatively unique alleles present in the coastal population, supporting its importance in the context of maintaining the remaining genetic variability of jaguars in the AF. Our results highlight the urgent need to implement coordinated conservation actions for jaguars in this highly impacted biome, including management interventions that foster the restoration of genetic connectivity among isolated remnant populations.