Hybridization and introgression are common in plants and lead to morphological similarity between species and taxonomic confusion. This gene flow with closely related species can complicate efforts to determine whether an endangered taxon is evolutionarily distinctive and should be identified as a separate conservation unit. Potentilla delphinensis is a rare and threatened endemic species of the Southern French Alps. Two common related taxa (P. grandiflora and P. thuringiaca) are morphologically similar and occur in the same geographical locations. Thus, whether P. delphinensis represents a reliable conservation unit remained unclear. Our evaluation procedure based on a combination of molecular biology and interspecific crosses was used to define taxa within these plants. Plants were sampled from a total of 23 single and mixed localities for the three supposed taxa and were genotyped with 68 polymorphic Amplified Fragment Length Polymorphism (AFLP) loci. Fourty-one seedlings from interspecific crosses were obtained and genotyped. Amplified Fragment Length Polymorphism markers identified four genetically distinct units (P. delphinensis, P. grandiflora and two distinct groups of P. thuringiaca). All individuals of P. delphinensis formed a homogeneous and distinct taxon. This taxon was most probably an old allopolyploid from P. grandiflora and the related group of P. thuringiaca. Interspecific crosses gave low seed set and low germination rate. Furthermore, assignment test indicated that seedlings obtained from interspecific crosses were essentially apomictic rather than hybrids. These results suggest that a reproductive barrier exists between the different taxa. In conclusion, all results supported P. delphinensis as a true biological species and justified its conservation unit status. A surprising outcome of this work was the evidence of a potential new cryptic species. This study demonstrated the need to combine a molecular marker-based approach and pollination experiments for an accurate evaluation of plant taxa. © 2007 Springer Science+Business Media, Inc.