AbstractThe study of ichthyoplankton is paramount to understanding fish assemblages' reproductive dynamics. DNA metabarcoding has been applied as a rapid, cost‐effective, and accurate taxonomy tool, allowing the identification of multiple individuals simultaneously. However, there remain significant challenges when using DNA metabarcoding, such as molecular marker choice according to the taxonomic resolution and length of the fragment to be sequenced, primer bias, incomplete reference databases, and qualitative inference incongruences. Here, 30 ichthyoplankton pools collected from a Neotropical river were identified at a molecular level using DNA metabarcoding to compare the resolution, sensibility, specificity, and relative read abundance (RRA) recovery of three molecular markers: the standard COI fragment (650 pb, with each end analyzed individually) and two short 12S rRNA genes markers (≅200 bp – NeoFish and MiFish markers). The combined use of the three markers increased the genera detection rates by 25%–87.5%, allowing an increased taxonomic coverage and robust taxonomic identification of complex Neotropical ichthyoplankton communities. RRA is marker‐dependent, indicating caution is still needed while inferring species abundance based on DNA metabarcoding data when using PCR‐dependent protocols.