Information on genetic aspects of jaguar populations is still scarce. Initial studies have surveyed genetic diversity parameters and assessed the geographic differentiation among individuals on a continental or sub-continental scale, but so far little has been accomplished with respect to investigating regional or local jaguar populations. Moreover, different studies have employed different sets of molecular markers, posing potential problems for the future development of comparative analyses across study sites and ecosystems. Here we review the current status of jaguar genetic studies, present a new set of microsatellite markers that may be useful for jaguar population genetic studies, and survey the molecular diversity of two adjacent wild jaguar populations, sampled in the Brazilian Pantanal region. Our results suggest that this set of markers is highly efficient for jaguar genetic studies, and that moderate to high levels of variability are present in wild jaguar populations, at least in the surveyed areas of the Pantanal. This contribution may be useful as a review of jaguar genetics, as well as a baseline empirical work that might support future in-depth investigations of these and other free-ranging populations of this felid.