In bats, sexual segregation has been described in relation to differential use of roosting and foraging habitats. It is possible that variation may also exist between genders in the use of different prey types. However, until recently this idea was difficult to test owing to poorly resolved taxonomy of dietary studies. Here, we use high-throughput sequencing to describe gender-related variation in diet composition of the European free-tailed bat (Tadarida teniotis), while controlling for effects of age and season. We analysed guano pellets collected from 143 individuals mist-netted from April to October 2012 and 2013, in northeast Portugal. Moths (Lepidoptera; mainly Noctuidae and Geometridae) were by far the most frequently recorded prey, occurring in nearly all samples and accounting for 96 out of 115 prey taxa. There were significant dietary differences between males and females, irrespective of age and season. Compared to males, females tended to consume larger moths and more moths of migratory behaviour (e.g. Autographa gamma). Our study provides the first example of gender-related dietary variation in bats, illustrating the value of novel molecular tools for revealing intraspecific variation in food resource use in bats and other insectivores.