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Tomini Bay in Gorontalo offers significant potential for amphidromous goby-fry, a group of fish found in at least five estuaries in the area, namely Bone-Bolango, Paguyaman, Bilungala, Tombulilato, and Taludaa Estuaries. Preliminary results were limited to only two locations in estuarine waters, namely the Bone-Bolango and Paguyaman rivers. Further exploration of goby-fry species in various locations within Tomini Bay is essential to enhance knowledge about the abundance and aid conservation efforts. Therefore, this study identified the goby-fry species and created their distribution map in the waters of Tomini Bay Gorontalo. The samples used were obtained from daily catches of fishermen in the five estuaries during the recruitment period from February to September 2022. These samples were categorized based on their morphological similarities and specific melanophore patterns. Distinct groups with different melanophore patterns from those previously reported were identified as newly recorded species, photographed, and described in terms of their morphology. Furthermore, two specimens from each newly recorded species underwent molecular identification using the cytochrome oxidase subunit 1 (COI) gene for DNA amplification and were analyzed through the Basic Local Alignment Search Tool (BLAST) method. The phylogenetic tree was constructed using the Maximum Likelihood Method. The results showed the existence of nongoby-fry species caught together with goby fry school. A total of 75,881 goby-fry and 1,687 nongoby-fry were successfully collected. Among the goby-fry species, 13 were identified, including three new records, namely Eleotris fusca (Forster, 1801), Sicyopterus microcephalus (Bleeker, 1855), and Sicyopus zosterophorus (Bleeker, 1856). This study also documented the existence of nongoby-fry species, namely Anguilla celebesensis (Kaup, 1856), Moringua microchir (Bleeker, 1853), and Microphis leiaspis (Bleeker, 1854). It significantly contributed to the understanding of fish biodiversity in Tomini Bay.