Adult Barn Swallows Hirundo rustica exhibit strong sexual size dimorphism in the length of the outermost tail feathers, which are longer in males compared with females. This trait is traditionally used to sex adult Barn Swallows in the field. However, due to the wear and breakage of the tips of the outer tail feather and tail moult during the non-breeding season, sexing becomes unreliable or even impossible. We therefore tested whether the length of the white spot on the outer tail feather is sexually dimorphic, and whether it can be used as an additional sexing method for adult Barn Swallows. The white spot length was sexually dimorphic, based on DNA analysis of 101 adult individuals caught at their roost during the non-breeding season in Zambia. Accuracy in sex determination of 95% could be obtained by classifying individuals with a white spot length 29.5 mm as males. When applying the length of the white spot as an additional method to sex adult Barn Swallows on all birds caught in Zambia during the study period (N = 759), the percentage of birds that could successfully be sexed increased to more than 55%. Therefore we emphasise the importance of measuring the white spot length in addition to the tail fork depth and tail length to sex adult Barn Swallows in the non-breeding season.