Sex identification is crucial for behavioral, ecological and conservation studies. In monomorphic bird species, traditional methods for sex assessment require potentially invasive sampling and manipulation of individuals, such as through cloacal examination. Thus, molecular methods involving non-destructive sampling are needed to reduce the stress of animals under study. In monomorphic Adélie penguins (Pygoscelis adeliae), the use of a penguin-specific primer pair (PL/PR) to identify sex has been evaluated through DNA extracted from nondestructive feather samples. With penguin-specific primers for the CHD1 gene found on the sex chromosomes, we efficiently identified the sex of all sampled individuals (n = 92, belonging to 46 complete nesting pairs in study colonies; n = 26 belonging to surveyed individuals in a control colony). DNA extraction and PCR protocols to improve amplification success are reported. Our results were also confirmed through Sanger sequencing used to determine the previously unpublished sequences of the CHD1 alleles in Adélie penguins, which were subsequently deposited in GenBank and used to compare to similar species. In phylogenetic reconstructions, CHD1Z and CHD1W sequences of the Adélie Penguin grouped together with other Pygoscelis and were the sister group of Spheniscus and Eudyptes genera.