Simple Summary Over 50% of birds are monomorphic, showing no sexually dimorphic traits, and in nestlings, the percentage is even higher. Early sex determination can be of great value in the management of wild birds, breeding of different bird species, improvement of breeding programs of captive birds, and evolutionary studies fields, and also for bird owners. In this study, we performed molecular sexing of wild and companion birds from various orders, such as Falconiformes, Accipitriformes, Galliformes, Anseriformes, Passeriformes, and Psittaciformes. Samples of oral swabs, feathers, and blood were collected from 43 birds. Conventional PCR was used to amplify the CHD1-Z and CHD1-W genes. The obtained results show that all types of samples can be used for molecular sexing of the studied species of birds. In conclusion, instead of using blood samples, our recommendation is to use minimally invasive samples (oral swabs and feathers) and test both types of samples on each bird.Abstract Birds are highly social and must be paired in order to increase their welfare. Most bird species are monomorphic; therefore, molecular sexing helps provide appropriate welfare for birds. Moreover, early sex determination can be of great value for bird owners. The aim of this study was to demonstrate that sex identification in birds achieved using molecular methods and samples collected via minimally invasive methods is fast, efficient, and accurate. A total of 100 samples (29 paired samples of feathers and oral swabs and 14 tripled samples of feathers, oral swabs, and blood) from 43 birds were included in this study, as follows: wild birds (Falconiformes, Accipitriformes, landfowl-Galliformes, waterfowl-Anseriformes) and companion birds (Passeriformes, Psittaciformes-large-, medium-, and small-sized parrots). Amplification of CHD1-Z and CHD1-W genes was performed via conventional PCR. The results obtained from feathers were compared to those obtained from oral swabs and to those obtained from blood samples, where applicable. The obtained results show that all types of samples can be used for molecular sexing of all studied bird species. To the best of our knowledge, the present study reports, for the first time, molecular sex identification in Red Siskin (Carduelis cucullata) and Goldfinch (Carduelis carduelis major). For higher accuracy, our recommendation is to use minimally invasive samples (oral swabs and feathers) and to test both types of samples for each bird instead of blood samples.
