Molecular and morphological data were used to evaluate the taxonomic status of the species Holothuria tubulosaGmelin, 1790, Holothuria stellatiDelle Chiaje, 1823, Holothuria mammataGrube, 1840, and Holothuria dakarensis Panning, 1939, belonging to the nominate subgenus Holothuria (Holothuria) (family Holothuriidae) from the Mediterranean Sea and Atlantic Ocean. A 16S rRNA marker distinguished three well-supported clades with clear genetic differentiation amongst them. The morphometric characters, although they reflected the clades, showed great variability, and some specimens from different clades overlapped. The morphological data and the literature suggest that the clades correspond to H. dakarensis (from Cape Verde Islands), H. mammata (from the Atlanto-Mediterranean area) and H. tubulosa (from the Mediterranean Sea). Holothuria stellati is considered here to be a junior subjective synonym of H. tubulosa. Great morphological intraspecific variation within H. tubulosa and H. mammata explains the confusion in the literature. Holothuria tubulosa includes specimens with distinctive ossicles, but others are similar to H. mammata. In these cases, the presence or absence of Cuvierian tubules proved a reliable indicator to the identity of these species; unfortunately this character is difficult to assess in preserved material. According to the results of discriminant analysis we propose a set of ossicle morphometric variables that permit the optimum assignation of individuals to the clades. Our results present a new perspective on the taxonomic status of species in Holothuria (Holothuria), and show how a molecular approach, combined with a morphological approach, can solve taxonomic problems.