<p>One of the steepest reductions in jaguar distribution in the Americas is occurring at the southern end of its range, in Argentina. The species has disappeared from the central portion of the country and is currently confined to three northern ecoregions: the Yungas, the Chaco and the Green Corridor of the Upper Paraná Atlantic Forest. Using nine microsatellite loci we characterise the genetic diversity and structure of these three jaguar populations (<i>n</i> = 54). We also analyse our data in conjunction with those previously reported for Amazonian, Pantanal and other Atlantic Forest populations (<i>n</i> = 255). Argentine jaguars are structured into two genetic groups: Chaco+Yungas and the Green Corridor of the Upper Paraná Atlantic Forest. Both groups show similar genetic diversity, which is lower than that reported elsewhere in South America. On a continental scale, jaguars from Chaco and Yungas were grouped along with individuals from the Amazon and northern Pantanal, while Upper Paraná Atlantic Forest jaguars were assigned to the coastal Atlantic forest population. We associate the observed genetic pattern with a strong signal of local genetic drift, coupled with isolation by distance and little or no gene flow between populations. In this context, it is essential that the increasing ecological disconnection between the Chaco and Yungas ecoregions caused by landscape alterations does not become a genetic disconnection. The isolation of the Upper Paraná Atlantic Forest population is particularly worrying because is isolated from both other Atlantic Forest and Chaco+Yungas populations. Our results emphasize the urgent need for management actions at the regional level, taking into account Jaguar Conservation Units and Corridors.</p>