Spider venom composition typically differs between sexes. This pattern is anecdotally thought to reflect differences in adult feeding biology. We used a phylogenetic approach to compare intersexual venom dimorphism between species that differ in adult niche dimorphism. Male and female venoms were compared within and between related species of Hawaiian Tetragnatha, a mainland congener, and outgroups. In some species of Hawaiian Tetragnatha adult females spin orb-webs and adult males capture prey while wandering, while in other species both males and females capture prey by wandering. We predicted that, if venom sexual dimorphism is primarily explained by differences in adult feeding biology, species in which both sexes forage by wandering would have monomorphic venoms or venoms with reduced dimorphism relative to species with different adult feeding biology. However, we found striking sexual dimorphism in venoms of both wandering and orb-weaving Tetragnatha species with males having high molecular weight components in their venoms that were absent in females, and a reduced concentration of low molecular weight components relative to females. Intersexual differences in venom composition within Tetragnatha were significantly larger than in non-Tetragnatha species. Diet composition was not different between sexes. This striking venom dimorphism is not easily explained by differences in feeding ecology or behavior. Rather, we hypothesize that the dimorphism reflects male-specific components that play a role in mating biology possibly in sexual stimulation, nuptial gifts and/or mate recognition.