Determination of sex in birds is valuable for studying population dynamics and structure, habitat use, behavior and mating systems. The purpose of the present study was to optimize a DNA-based methodology to allow the sex identification in Accipiter cooperii nestlings. Chromo-helicase-DNA-binding (CHD1) gene was used in this work as a marker for sex identification. CHD-W and CHD-Z sequences should present length and/or sequence differences providing a way to identify gender. We used a non-invasive method for DNA extraction from feathers and performed polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) method. The length difference between CHD-W and CHD-Z amplified fragments observed by electrophoresis in conventional agarose gel was not enough to provide a clear differentiation between males and females. However, patterns obtained by PCR-SSCP differentiated undoubtedly males and females in A. cooperii. This tool provides a precise gender identification assay and will be applied to confirm and refine morphometrically based sexing techniques used in the field.