DNA barcoding is a widely used tool for species identification, with its reliability heavily dependent on reference databases. While the quality of these databases has long been debated, a critical knowledge gap remains in their comprehensive evaluation and comparison at regional scales. Marine metazoan species in the western and central Pacific Ocean (WCPO), a region characterized by high biodiversity and limited sequencing efforts, are an example of this gap. This study developed a systematic workflow to assess mitochondrial cytochrome c oxidase subunit I (COI) barcode coverage and sequence quality in two commonly used reference databases for DNA barcoding: the nucleotide reference database from the National Center for Biotechnology Information (NCBI); and from the Barcode of Life Data System (BOLD). Comparative analyses across marine phyla and WCPO regions identified significant barcode gaps and quality problems, providing insights to guide future barcoding efforts. NCBI exhibited higher barcode coverage, but lower sequence quality compared to BOLD. Quality issues, including over- or under-represented species, short sequences, ambiguous nucleotides, incomplete taxonomic information, conflict records, high intraspecific distances, and low inter-specific distances were identified in both databases, likely resulting from contamination, cryptic species, sequencing errors, or inconsistent taxonomic assignment. The barcode identification number (BIN) system in BOLD demonstrated potential for identifying and addressing problematic records, highlighting the benefits of curated databases. Significant barcode deficiencies and quality issues were observed in the south temperate region of WCPO and phyla such as Porifera, Bryozoa, and Platyhelminthes. Additionally, the COI barcode showed limited species-level resolution for certain taxa, including Scombridae and Lutjanidae. Addressing barcode coverage gaps, improving taxonomic representation, and enhancing sequence quality will be essential for strengthening future barcoding initiatives and advancing biodiversity monitoring and conservation in the WCPO and beyond. This study highlights the need for standardized database curation and sequencing practices to improve the global reliability and applicability of DNA barcoding.