During secondary contact between phylogenetically closely related species (sibling species) having diverged in allopatry, the maintenance of species integrity depends on intrinsic and extrinsic reproductive barriers. In kelps (Phaeophyceae), the observations of hybrids in laboratory conditions suggest that reproductive isolation is incomplete. However, not all interspecific crosses are successful, and very few hybrids have been observed in nature, despite the co-occurrence of many kelp species in sympatry. This suggests that there are reproductive barriers that maintain species integrity. In this study, we characterized the fine genetic structure of a secondary contact zone to clarify the extent of reproductive isolation between two sister species. In Lessonia nigrescens Bory (Laminariales, Phaeophyta) species complex, two cryptic species have been recently found out from gene phylogenies, and-waiting for a formal taxonomic description-we used their geographic distribution to name them (northern and southern species). We studied 12 populations, distributed along 50km of coastline, and employed two molecular approaches, assigning individuals to phylogenetic species according to a diagnostic mitochondrial marker (351 individuals analyzed) and quantifying interspecific gene flow with four microsatellite markers (248 individuals analyzed). No hybridization or introgression was revealed, indicating complete reproductive isolation in natural conditions. Unexpectedly, our study demonstrated that the two species were strictly segregated in space. This absence of co-occurrence along the contact zone can partially explain the lack of hybridization, raising new interesting questions as to the mechanisms that limit sympatry at small spatial scales. © 2011 Phycological Society of America.