Most species of penguins are sexual monomorphic and therefore it is difficult to visually identify their genders for monitoring population stability in terms of sex ratio analysis. In this study, we evaluated the suitability using melting curve analysis (MCA) for high-throughput gender identification of penguins. Preliminary test indicated that the Griffiths's P2/P8 primers were not suitable for MCA analysis. Based on sequence alignment of Chromo-Helicase-DNA binding protein (CHD)-WandCHD-Zgenes from four species of penguins (Pygoscelis papua, Aptenodytes patagonicus, Spheniscus magellanicus, andEudyptes chrysocome), we redesigned forward primers for theCHD-W/CHD-Z-common region (PGU-ZW2) and theCHD-W-specific region (PGU-W2) to be used in combination with the reverse Griffiths's P2 primer. When tested withP. papuasamples, PCR using P2/PGU-ZW2 and P2/PGU-W2 primer sets generated two amplicons of 148- and 356-bp, respectively, which were easily resolved in 1.5% agarose gels. MCA analysis indicated the melting temperature (Tm) values for P2/PGU-ZW2 and P2/PGU-W2 amplicons ofP. papuasamples were 79.75°C–80.5°C and 81.0°C–81.5°C, respectively. Females displayed both ZW-common and W-specific Tm peaks, whereas male was positive only for ZW-common peak. Taken together, our redesigned primers coupled with MCA analysis allows precise high throughput gender identification forP. papua, and potentially for other penguin species such asA. patagonicus, S. magellanicus, andE. chrysocomeas well. [ABSTRACT FROM AUTHOR]