Consistent, repeatable segregation of plant species along hydrological gradients is an established phenomenon that must in some way reflect a trade-off between plants’ abilities to tolerate the opposing constraints of drought and waterlogging. In C3 species tissue carbon isotope discrimination (d13C) is known to vary sensitively in response to stomatal behaviour, reflecting stomatal limitation of photosynthesis during the period of active growth. However, this has not been studied at fine-spatial scale in natural communities.We tested how d13C varied between species and sexes of individuals in the family Restionaceae growing along a monitored hydrological gradient. Twenty Restionaceae species were investigated using species-level phylogeny at two sites in the Cape Floristic Region, a biodiversity hotspot. A spatial overlap analysis showed the Restionaceae species segregated significantly (P < 0.001) at both sites. Moreover, there were significant differences in d13C values among the Restionaceae species (P < 0.001) and between male and female individuals of each species (P < 0.01). However, after accounting for phylogeny, species d13C values did not show any significant correlation with the hydrological gradient.We suggest that some other variable (e.g. plant phenology) could be responsible for masking a simple response to water availability.