Endemic Florida Mottled Duck (Anas fulvigula fulvigula) appear to be hybridizing introgressively with domesticated Mallards (A. platyrhynchos), which are frequently released or escape captivity and have established feral populations throughout Florida. To investigate this possible conservation threat, we isolated and characterized 59 polymorphic loci from an enriched Florida Mottled Duck microsatellite library and performed cross-amplification assays with Mallard specimens. Average numbers of alleles per locus were 6.0 (ranging 2-23) and 5.6 (ranging 2-15) for A. fulvigula and A. platyrhynchos, respectively; estimates of observed/expected heterozygosity were 0.54/0.63 and 0.52/0.64. Markers developed in this study will be used in conjunction with existing markers to robustly classify hybrids and to assess and monitor the genetic dynamics of introgression between these waterfowl species.