Pterodroma phaeopygia is a critically endangered avian species of the Galápagos Islands. This bird is sexually monomorphic, making it difficult to identify the sex. This information, however, is relevant to studies of behavior, ecology, and management of wild or captive populations. Here, we aimed to implement a molecular approach for determining sex in this petrel. DNA was extracted from the blood and the feathers of 24 adult P. phaeopygia, with samples from a female and a male Gallus gallus for comparison. We amplified the cromo-helicase DNA binding protein 1 (CHD-1) gene by PCR, using primers P2 and P8. Allele CHD-1W is unique to females and CHD-1Z occurs in both sexes. We then digested these PCR products using the restriction enzyme HaeIII. The PCR amplified a 400-bp product for both alleles. The digestion of the G. gallus, amplicons split the CHD-1Z allele into two fragments (of 320 and 80 bp), while CHD-1Wremained intact. Thus, the male exhibited two bands (digested CHD- 1Z) and the female three bands (undigested CHD-1W and digested CHD-1Z). Applying this RFLP method on DNA derived from blood, 9 of the 24 petrels were found to be male, while 15 were females. The same results were obtained using feathers as the source of DNA. To our knowledge, this is the first report of molecular method for sexing this species. The potential of sexing this petrel from feathers is remarkable as it minimizes blood sampling induced stress. This method could be used to reinforce the conservation efforts for this bird, to investigate population sex ratios and to develop new conservation strategies.