The processes underlying reproductive isolation, and the traits involved, are the subject of considerable debate in evolutionary biology. Studying the costly sexual interaction of species in secondary sympatry, a phenomenon known as reproductive interference, can help to shed light on past and present isolating mechanisms, as well as the implications of sympatry for individual fitness. We investigated the roles of two sets of traits, genitalic and behavioural, in reproductive isolation and interference in two species of Australian neriid flies, Telostylinus lineolatus and T. angusticollis. Surprisingly, although these species differ markedly in male but not in female genitalia, we found evidence that genital morphology resulted in asymmetric reproductive isolation: T. lineolatus males could transfer sperm to T. angusticollis females, but T. angusticollis males were unable to transfer sperm to T. lineolatus females. However, neither type of cross produced any viable hybrids. Behavioural responses also contributed asymmetrically to both reproductive isolation and reproductive interference. Males pursued both conspecific and heterospecific females. Females of both species discriminated against heterospecific males, but T. lineolatus females exhibited stronger discrimination than T. angusticollis females. Curiously, T. angusticollis males both fought and attempted to copulate with T. lineolatus males, resulting in reduced mating success for T. angusticollis males with conspecific females. Our findings show that both genitalic and behavioural traits can play important roles in reproductive isolation, but the consequences of interspecific divergence in these traits can be surprisingly complex, resulting in asymmetric effects on many aspects of interspecific interactions.