The ecological niche defines the favourable range of a species in a multidimensional space of ecological factors that determine the presence and function of individuals. This fundamental concept in ecology is widely used to understand plant species coexistence and segregation. In this study, we test for ecological differentiation among six annual Lythrum species that are characteristic of temporary pools in the South of France, where they either coexist or occur separately. We first analysed the co-occurrence of species at two different geographical scales: cluster analyses of species presence in 10 km grid cells and coexistence in 0.25 m2 quadrats within populations of each species. Second, for three to nine populations of each species, we quantified a range of biotic and abiotic parameters using point contacts and soil measurements in five 0.25 m2 quadrats per population. We performed PCA on all variables and analysed each variable separately to compare the ecological niche features of the six species. A phenological index was assessed for the plant community of each site. We detected highly localized niche differentiation in terms of soil pH (all species) and for a range of variables among pairs of species. The six species also showed marked differences in the flowering period relative to the mean and variability of flowering time in their local community. These fine-scaled niche differences are associated with phylogenetic distances among species and may contribute to species’ coexistence. These results are integrated in a conservation management plan for the habitat of the rarest species in this group.