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Abstract The Mallard (Anas platyrhynchos) duck is a ubiquitous and socio-economically important game bird in North America. Despite their generally abundant midcontinent population, Mallards in eastern North America are declining, which may be partially explained by extensive hybridization with human-released domestically derived game-farm Mallards. We investigated the genetic composition of Mallards in the middle and lower Mississippi flyway, key wintering regions for the species. We found that nearly 30% of wild Mallards carried mitochondrial (mtDNA) haplotypes derived from domestic Mallards present in North America, indicating that the individuals had female game-farm Mallard lineage in their past; however, nuclear results identified only 4% of the same sample set as putative hybrids. Recovering 30% of samples with Old World (OW) A mtDNA haplotypes is concordant with general trends across the Mississippi flyway and this percentage was stable across Mallards we sampled a decade apart. The capture and perpetuation of OW A mtDNA haplotypes are likely due to female breeding structure, whereas reversal of the nuclear signal back to wild ancestry is due to sequential backcrossing and lower and/or declining admixture with game-farm Mallards. Future studies of wild ancestry of Mississippi flyway Mallards will benefit from coupling molecular and spatial technology across flyways, seasons, and years to search for potential transitions of Mallard populations with different genetic ancestry, and whether the genetic ancestry is somehow linked to an individual’s natal and subsequent breeding location.