Integrating biological, morphological and molecular data is necessary for the phylogeny of Platyhelminthes. Besides the molecular tools, the ultrastructural description of the mature spermatozoon was found a valuable tool for this purpose. Among the studied cestodes, description of Hymenolepis diminuta spermatozoon has been neglected; therefore, this study was designed. Live specimens of Hymenolepis spp. were isolated from naturally infected rats and molecular sequencing of the internal transcribed spacer (ITS2) region of the major ribosomal RNA genes was performed and a phylogenetic tree was constructed. This analysis revealed a 99–100% identity of the isolated worm as H. diminuta which appeared distinctive in a subclade while H. nana and H. microstoma appeared more closely related in another subclade. After the
molecular identification of the worm, an ultrastructure study was performed. This might be the first ultrastructure characterization of a parasitic Platyhelminth spermatozoon in which a molecular identification tool was utilized. The results revealed a filiform cell with a single axoneme of 9 + “1" pattern extending from the anterior to the posterior extremity, a spiraled cordlike nucleus situated posteriorly associated with a large amount of glycogen, spiraled cortical microtubules at an angle of 20-25º then they became parallel to the axoneme at the posterior extremity. For the first time, this report six crested-like bodies at the anterior extremity of H. diminuta mature spermatozoon. These crested bodies enabled to clarify spermatozoon orientation and provided a full schematic reconstruction of it.