Accurate information of taxonomy and geographic range of seahorse species (genus Hippocampus) is the first step in preparing threat assessments and designing effective conservation measures. Here, we report first records and a range extension of the Japanese seahorse, Hippocampus mohnikei (Bleeker, 1853) from the Mandovi estuarine ecosystem of Goa, central west coast of India (CWCI) based on morphological and molecular analyses. The morphometric and meristic traits, particularly short snout (29-35% head length), double cheek spine, low coronet, long tail (51.2-57.9% of standard length), 11 trunk rings, 37-39 tail rings, 15-16 dorsal and 12-14 fin rays observed in four collected specimens matched with the reported key diagnostic morphological criteria of vouchered specimens of H. mohnikei. The seahorse mitochondrial cytochrome oxidase subunit I (COI) and cytochrome b (Cyt b) genes were partially sequenced for conclusive genetic identification of the species under study. Molecular analysis showed that all four individuals clustered together suggesting a monophyletic lineage. Using the maximum similarity with GenBank database, maximum likelihood network and subsequent morphological analysis, the identity of the collected seahorse species was reconfirmed as H. mohnikei. With this new report, the geographic range of H. mohnikei extended significantly to the west from its previously known range. This new sighting of H. mohnikei could indicate a long-distance dispersal facilitated by the prevailing oceanic circulation in the Indo-Pacific region or increased habitat suitability in bay-estuarine systems of Goa, CWCI. Comparison of the pair-wise genetic distances (Kimura 2-parameter) based on COI and Cyt b sequences revealed that the specimens examined in this study are genetically closer to H. mohnikei populations from Vietnam and Thailand than they are to those in Japan and China. To test the hypothesis whether H. mohnikei are vagrants or previously unreported established population, long-term inter-annual sampling and analyses are warranted. PLOS ONE PLOS ONE | https://doi.org/10.1371/journal.pone.0220420 March 24, 2020 1 / 22 OPEN ACCESS Citation: Sanaye SV, Khandeparker R, Rayadurga AS, Shivaramu MS, Kankonkar H, Narvekar J, et al. (2020) Morphological and molecular evidence for first records and range extension of the Japanese seahorse, Hippocampus mohnikei (Bleeker 1853) in a bay-estuarine system of Goa, central west coast of India. PLoS ONE 15(3): e0220420. https://