Development of a reliable PCR‐based protocol for molecular sexing of king vultures Sarcoramphus papa

The king vulture Sarcoramphus papa is a large Neotropical scavenger that lacks external sexual dimorphism, complicating demographic, ecological, and conservation studies. Reliable sex identification is therefore essential, particularly for wild and managed populations. In this study, we assessed and optimized a PCR‐based molecular sexing protocol for S. papa using amplification of the sex‐linked chromodomain helicase DNA binding 1 (CHD1) gene. Samples from eight wild individuals captured in western Ecuador were analyzed using three biological sample types – whole blood, feathers, and blood preserved on Whatman FTA cards – and three commonly used CHD1 primer sets. All primer sets successfully amplified CHD1 fragments; however, amplification consistency, band clarity, and ease of interpretation varied among sample types and primers. The primer pair 2550F/2718R produced the most reliable and unambiguous results across all sample types, with a clear size difference (~ 750 bp) between CHD1‐Z and CHD1‐W fragments, allowing straightforward sex determination. This optimized protocol provides a robust and cost‐effective tool for molecular sexing of S. papa using multiple sample types, including blood, feathers, and blood preserved on Whatman FTA cards, and can support population monitoring, ecological research, and conservation management of this monomorphic species and other birds.