In many avian species, it is difficult to distinguish the sexes based on their external features. Sex determination is an important area of study in developmental and evolutionary biology, as well as in ecology. In penguins, physical sexual characteristics between males and females are hard to differentiate particularly the Jackass penguins (Spheniscus demersus) which show little sexual dimorphism. Although it is always thought that males are usually larger than females, sexing by direct observation may be difficult especially in young birds. In this research, we evaluated a sex determination technique in Jackass Penguins using genomic deoxyribonucleic acid (DNA) amplification using primers. Genomic DNA was extracted from feathers of Jackass penguins, Chickens (Gallus gallus domesticus) and Ducks (Anas Platyrhynchos var. domestica) using E prep (Viagenkorea.CO, Korea) reagent. A primer set is designed for sex determination. The primers amplifythe homologous region of the CHD-W gene (chromo-helicase-DNA-binding gene) unique to females, and the CHD-Z gene, occurring in both sexes and characterized by two bands in females and only one band in males. The results showed that analyses of the polymerase chain reaction products in Jackass penguins using 2550F-2718R and P2/P8 primer sets showed one band in both males and females. Moreover, 2550F-2718R primer set identified the sex of chickens but not in ducks while primer P2/P8 showed only one band in both chickens and ducks. The 1272L-1237H primer showed one band in male and two bands in female in all species tested. It was confirmed that the use of 1272L-1237H primer concur in identifying the sex of the following species: Jackass penguins, ducks and chickens.