We report an unusual and large-sized Ophioglossum plants (up to 32 cm.), as O. aletum sp. nov., found on forest plains in Southern Gujarat, which are characterized by possessing almost 90% alete spores mostly growing in chains; a feature never recorded in any species in the world flora of the genus. Alete spores are known but in too-low a frequency (less than 0.5%) in many species found in India. These plants, designated as O. aletum sp. nov. resemble O. vulgatum L. in leaf morphology and O. eliminatum Khandelwal & Goswami in exine ornamentation of spores under SEM. But these plants differ from all species of Ophioglossum so far known in the botanical literature in possessing larger alete spores (up to 44 µm) developing mostly as linear ‘tetrads’ and very long petiole showing periderm. This has been repeatedly confirmed that spore mother cells after first division separate as dyads and then each cell matures and separates as a spore, hence the spores do not develop any proper “lete”. We have observed series of linear ‘tetrads’, grouped spores and even randomly distributed double and single spores. The proximal face of each bowl-shaped spore has thicker lining on the round margin of spore suggesting that spores were separated and developed from dyad, before reaching a ‘tetrad phase’. Such developmental phases have never been observed so far in any species of Ophioglossum. Molecular study based on rbcL chloroplast DNA sequences supports O. aletum sp. nov. as a distinct species, which nested within O. vulgatum clade and is closest to O. petiolatum. Genetically, the new species differs from O. eliminatum and O. vulgatum in an uncorrected pair-wise sequence divergence of 8.37% and 5.43% respectively for a fragment of chloroplast ribulose-1, 5-bisphosphate carboxylase (rbcL) gene.