<p>The accurate assessment of ichthyoplankton (fish eggs and larvae) diversity is frequently impeded by their small size and complex morphology. To enhance taxonomic resolution, we utilized a rigorous in silico pipeline to design and confirm the efficacy of two novel primer sets targeting the cytochrome c xidase subunit I (COI) gene. The first set, universal primers (UP), demonstrated broad taxonomic utility, achieving up to 90% successful amplification in target phyla and 75% in nontarget phyla. The second set, Chordata-specific primers (CSP), was engineered for precise targeting of this phylum, and successfully applied to an ichthyoplankton case study in Vietnam, yielding a high amplification rate of 94.41%. Comparison with traditional morphological methods showed that molecular identification, enabled by these optimized markers, increased the capacity for species detection, especially for specimens with underdeveloped features. While incomplete reference databases occasionally constrained this resolution, the study strongly advocates for the integration of genetic and morphological approaches for robust species assessment. These new COI markers offer a powerful resource for biodiversity research in complex ecosystems, holding substantial implications for conservation and fisheries management.</p>