Morphometric methods of sex differentiation may be cheap, simple, quick, and reliable alternatives to molecular approaches. However, there are still important uncertainties regarding the use of morphometric methods in birds, particularly regarding their applicability to different populations and environmental conditions. Between 2004 and 2019, we sampled 245 Bonelli´s Eagle Aquila fasciata nestlings, 197 from nests in Catalonia (northeastern Spain) and Andalusia (southeastern Spain) and 56 that were partially or totally raised in captivity. Our objective was to develop morphometry-based sexing methods for Bonelli’s Eagle nestlings that can be applied in situ to different subpopulations and growth conditions. We recorded up to nine measures related to nestling body mass, bill, tarsus, and claw length. Tail and wing length were used to control for the age of nestlings, and all individuals were sexed genetically. We found important morphometric differences between the two natural subpopulations. According to our discriminant analyses, body mass and claw length were the most discriminant variables, both in wild and captivity conditions. Differences between sexes were more prominent during the flight feathers’ growing period. Feeding ad libitum in captivity conditions led to larger nestlings and reduced between-sexes differences. We provide different models for sexing Bonelli’s Eagle nestlings that can be applied according to the studied geographic location, growth conditions (wild versus captive), and balance between accuracy and simplicity. Overall, our findings highlight that extrapolating sex discriminant functions to different subpopulations and growth conditions may be risky.