Triterpene glycosides of three species of the Mediterranean sea cucumbers Holothuria polii, Holothuria tubulosa, and Holothuria sp. were studied. Three new monosulfated biosides, holothurins B(2) (1), B(3) (2), and B(4) (3), along with the previously known holothurins A (4) and B (5) were isolated from the sea cucumber H. polii. Triterpene glycosides belonging to holothurin A and B groups were found in H. tubulosa, while only one individual glycoside, holothurin A (4), was isolated from Holothuria sp. Structures of new substances were elucidated on the basis of spectral data (2D NMR and MS). The significance of holothurins as chemotaxonomic markers of the animals belonging to the genus Holothuria and taxonomic status of some representatives of the holothurians studied are discussed.