The global fish industry has been the fastest-growing food production sector over the last two decades and is expected to continue growing due to the increasing demand for seafood worldwide. Trailing the rapid expansion of fish production and consumption is the unfortunate consequential rise of fish mislabelling, which is the deliberate or accidental substitution of one fish species with another. Mislabelling can negatively impact consumer finances, consumer health and endanger the survival of certain fish species. DNA barcoding is the use of DNA sequences for genetic identification and has been used to uncover widespread mislabelling in many countries. This study is the first to investigate the extent of fish mislabelling in Singapore, using novel degenerate primers for the Cytochrome c oxidase subunit I (COI) gene. A total of 106 DNA samples were extracted from raw, processed and cooked fish samples collected from eateries, supermarkets and wholesale markets across Singapore. Amplification and sequencing of the COI gene sequences were followed by interrogation of the National Centre for Biotechnology Information (NCBI) database using Basic Local Alignment Search Tool (BLAST). Fish identity was then checked against the FishBase Information System to determine whether labelled fish names had been incorrectly assigned. Out of 106 fish samples, 105 samples were successfully amplified, sequenced and identified. Among those samples, 21 (20%) were found to be mislabelled. Of the mislabelled fish, Grouper (38.5%) and Snapper (36.4%) were the most commonly mislabelled and surprisingly, none of the 20 tuna samples were found to be mislabelled. Three of 19 salmon samples (15.8%) were found to be Rainbow trout. This study confirms that fish mislabelling is prevalent in Singapore, which is on par with the global average, and consumer finances and health are of genuine concern to people who place trust in the purchase their fish products.