Centric diatoms of the genus Leptocylindrus are common in the marine plankton worldwide. Only two species, L. danicus Cleve and L. minimus Gran, so far clearly belong to this genus, whose diversity has not been fully investigated. We investigated frustule and spore morphology as well as three nuclear- and three plastid-encoded markers of 85 Leptocylindrus strains from the Gulf of Naples, and one from the Atlantic US. The strains grouped into five molecularly distinct species with different levels of morphological differentiation. Two species matched the description of L. danicus and produced similar spores but differed in morphometric characters and sub-central pore position, supporting the description of L. hargravesii Nanjappa and Zingone as a distinct species. Leptocylindrus danicus var. apora French III and Hargraves, lacking a sub-central pore and not forming spores, was raised to the species level as L. aporus (French III and Hargraves) Nanjappa and Zingone. A fourth species with convex valves was described as L. convexus Nanjappa and Zingone. The fifth species matched the description of L. belgicus Meunier, considered as synonym of L. minimus. However, ultrastructural differences from all other Leptocylindrus supported the erection of the genus Tenuicylindrus Nanjappa and Zingone with T. belgicus (Meunier) Nanjappa and Zingone as type species. None of the sequences matched the L. minimus sequence in GenBank. The species analyzed showed different or partially overlapping seasonal distributions. Despite the addition of the new taxa, the ancient diatom lineage of the Leptocylindraceae shows a relative species poorness and considerable morphological stasis.