Background and aims: Ecological niche differentiation is well associated with intraspecific divergence of functional traits, which may lead to the evolution of premating reproductive isolation. However, the link between the ecological niches, trait divergence, and premating isolation remains poorly understood. This is particularly pertinent in hyper-diverse areas such as the Cape Floristic Region (CFR) of South Africa, where fine-scale ecological heterogeneity has been hypothesised as a major driver of speciation. Using the polymorphic geophyte Gladiolus carneus, endemic to the CFR, we test whether ecological niche differentiation mediates premating reproductive isolation. Methods: We first tested whether newly and previously described Gladiolus carneus varieties were distinct based on their floral and vegetative morphology. Next, we documented each varieties abiotic niche, flowering phenology and pollination niche and further tested whether any resulting niche differentiation causes premating reproductive isolation. Key results: Seven morphologically distinct varieties were identified, of which two of these are newly documented. Using niche modelling and multivariate analyses, we found that these varieties occupied distinct abiotic niches, resulting in strong ecogeographic isolation. They also had distinct flowering times, causing varying strengths of phenological isolation. For the pollinator niche, we found that all sampled populations had a single highly effective functional pollinator; however, at the variety level, there were no consistent trends leading to varying strengths in pollinator-mediated isolation. Across all varieties, ecogeographic isolation was the strongest gene flow barrier, which combined with phenological and pollinator-mediated isolation, causes near complete premating reproductive isolation. Conclusions: These results suggest that ecological niche differentiation between Gladiolus carneus varieties may be contributing to incipient speciation within the species complex and further suggests that ecological niche differentiation may be a major driver of speciation in the hyper-diverse Cape Floristic Region.