The great phenotypic variability and the lack of diagnostic characters in the genus Pylaiella render the systematic study of this genus problematic. In this study, we investigated the diversity of Pylaiella littoralis along the Brittany (France) coast using a DNA barcoding multilocus approach with mitochondrial (cox1, nad1, and atp9) and chloroplastic (rbcL and atpB) markers associated with a population genetics approach using 10 microsatellite markers. In addition, spatio-temporal sampling was conducted along the Brittany coast. We sampled 140 individuals from four sites located between Saint-Malo and Concarneau (380 km) from April to October. Mitochondrial sequence data revealed the occurrence of two sibling species, with a minimum of 2.4% divergence between them. Microsatellite genotypic data congruently revealed two well-supported clusters matching the two mitochondrial clades of Pylaiella. Although gene flow is limited between species, occurrence of genetic admixtures in some populations suggested that reproductive isolation is not complete. Our study highlighted the complementarity of barcoding and population genetics approaches to shed light on the evolutionary processes that lead to speciation.