Since there was a lack of study on the diversity and genetics of marine fishes in Pulau Tinggi, Mersing, Johor could be found to date, this study was conducted to provide a baseline data of species checklist of marine fishes in Pulau Tinggi and to obtain DNA barcodes of the fish specimens using cytochrome c oxidase subunit I (COI) gene. Sample collection in this study was conducted starting from July to August 2019. Spinning rod (angling), hand-net (manual sampling) and gillnet (mid-water column) were used as sampling methods in this study. All specimens obtained in this study were first identified based on morphological features, followed by a molecular approach for unidentified individuals. The morphological and molecular classification proved that all 183 individuals of marine fishes obtained in this study belonged to 83 species, 58 genera, 36 families, 9 orders and 2 classes. Among 83 species obtained in this study, 74 (89%) possess important values for human use. According to IUCN Red List online platform, 22 species are classified as Not Evaluated (NE), one species is classified as Data Deficient (DD), 56 species are classified as Least Concern (LC), three species are classified as Near Threatened (NT) and the remaining one species is classified as vulnerable (VU). A total of seven bidirectional CO1 barcode sequences were obtained from seven species, six genera and six families. All the sequences obtained have significant E values, which were zero, with percent identity values being more than 98% and query coverage values being more than 89%. The blast results for all the sequences provided correct identification until the species level. For future research, this study has updated the latest checklist of marine fishes from Pulau Tinggi, Mersing, Johor, Malaysia.