Identifying the sex of bird nestlings is relevant to studies of behaviour and ecology and is often a central issue in the management of endangered or captive populations. The white-tailed eagle Haliaeetus albicilla is a formerly threatened Eurasian raptor which is closely monitored in many countries due to its high exposure to environmental pollutants in the food chain. The aim of this study was to evaluate the reliability of sex identification methods for white-tailed eagle nestlings based on morphological measurements that can be recorded at the nest by a single person and with minimum disturbance. The sex of each bird was independently determined using molecular (genetic) methods. One measure of tarsus width allowed the correct identification of sex for 96% of the nestlings from southern and central Sweden. However, we found that the criteria for sex identification were not directly applicable to the population in Swedish Lapland, where nestlings are typically thinner, probably due to a limited food supply. These results show that sexing in the field of white-tailed eagle nestlings can be feasible with high accuracy based on a limited number of measurements. However, the criteria employed to separate sexes may have to be adjusted for each population.