Hawaiian ducks (Anas wyvilliana), or koloa, are endemic to the Hawaiian Islands and are listed as a federal and state endangered species. Hybridization between koloa and introduced mallards (A. platyrhynchos) is believed to be a primary threat to the recovery of koloa. We evaluated the utility of two sets of nuclear markers (microsatellite loci and amplified fragment length polymorphisms) and a variable portion of the mitochondrial DNA control region to distinguish among koloa, mallards, and hybrids. We show that microsatellite and AFLP markers can be used to distinguish between koloa and mallard-koloa hybrids with a high degree of confidence. For all but one of the putative koloa in our sample, the posterior probability of belonging to the koloa category was >0.90. Similarly all but one of the mallard-koloa hybrids were assigned to the hybrid category with posterior probabilities >0.98. Subsets of markers led to poorer resolution among koloa, mallard and hybrid categories. Among a sample of 61 koloa, hybrids and mallards, we found 25 different mtDNA haplotypes, belonging to two groups of haplotypes (A and B) identified previously in mallards and their relatives. All putative koloa samples exhibited group B haplotypes, of which 65% comprised one haplotype, while the rest were divided among four haplotypes. All Hawai’i mallard samples exhibited haplotypes that belonged to group A. Hybrids and California mallards exhibited haplotypes belonging to both groups, but a majority were of group A, suggesting that hybridization may more commonly involve mating between Hawai’i mallard females and koloa males.