Ophelia bicornis sensu lato is a polychaete living in intertidal sandy habitats of Mediterranean and European Atlantic coasts, whose systematics have been strongly debated in the past few decades. In the present work the count of nephridiopores was coupled with genetic analysis carried out with DNA markers (inter simple sequence repeats) for a total of 30 individuals collected at six Italian beaches. Exact test, analysis of molecular variance, non-metric multidimensional scaling and assignment tests clearly separated individuals with five nephridiopore pairs from those with six pairs. This finding validated results of a recent allozyme study in which O. bicornis sensu lato was split into O. bicornis sensu stricto (six nephridiopore pairs) and O. barquii (five nephridiopore pairs). This paper represents a further contribution to the estimation of biodiversity within marine invertebrates.