Sexing of barn owls, Tyto alba javanica, using morphological traits has not been accurate enough due to ambiguous sexual dimorphism between sexes. This has been one of the major problems for the management of barn owls worldwide, especially for translocation and captive-breeding programs. In order to increase the success rate of sexing the barn owl, we compared the results of a molecular sexing method to six morphological traits for sexing the owls: the shape and colour of the facial disc, the colour of the throat area, the tail plumage, the colour of their tarsus, the back plumage, and the frequency of spotting on the chest and underside of the wings. The result of our comparison showed that sex identification using morphological traits had an accuracy of only 72.7%. Three of our samples were identified as females using morphological traits, but molecular sexing determined that these samples were males. We also used our results to determine the best morphological traits for sexing barn owls, and concluded that the best traits for morphological sexing are the frequency of spotting on the chest and underparts of barn owls (accuracy of 81.8%), as well as colour of the owls’ facial disc and throat area (accuracy of 63.6%).