AbstractSexual segregation is widespread across the animal kingdom, yet there is limited consensus on the factors that shape this behavioural phenomenon. Many of the existing theories are based on study species with high levels of sexual size dimorphism. Insights from studies on species with minimal sexual size dimorphism, such as vespertilionid bats, provide an opportunity to study the factors associated with segregation irrespective of body size effects. Using long‐term data pooled from multiple bat box monitoring schemes across the U.K., we investigated segregation patterns in maternity roosts of three vespertilionid bat species, Bechstein’s bat, Myotis bechsteinii, Natterer’s bat, M. nattereri and brown long‐eared bat, Plecotus auritus. We used the Sexual Segregation and Aggregation Statistic (SSAS) to evaluate temporal trends of sexual segregation of roosts over the reproductive period (divided into pre‐parturition, lactation and post‐lactation periods). Additionally, we used generalized linear mixed models and beta regression models to investigate the effect of group size on segregation patterns. Our results showed that the size of the maternity group was an important covariate of inter‐ and intra‐sexual segregation, with males and non‐breeding females typically segregated from large maternity groups across all three periods. Additionally, we demonstrate that reproductive condition and period influence segregation patterns, with breeding females segregated from non‐breeding females and males during the lactation period. Although sexual segregation may be caused by multiple mechanisms, our results show that group size, female reproductive condition and time period are key factors associated with segregation within bat roosts. These findings make a valuable contribution to the understanding of inter‐ and intra‐sexual segregation in vespertilionid bats and complement existing research on segregation in other mammalian taxa, providing further evidence that sexual size dimorphism is not a prerequisite for sexual segregation.