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Abstract Metabarcoding of zooplankton communities is becoming more common, but molecular results must be interpreted carefully and validated with morphology-based analyses, where possible. To evaluate our metabarcoding approach within the California Current Ecosystem, we tested whether physical subsampling and PCR replication affects observed community composition; whether community composition resolved by metabarcoding is comparable to morphological analyses by digital imaging; and whether pH neutralization of ethanol with ammonium hydroxide affects molecular diversity. We found that (1) PCR replication was important to accurately resolve alpha diversity and that physical subsampling can decrease sensitivity to rare taxa; (2) there were significant correlations between relative read abundance and proportions of carbon biomass for most taxonomic groups analyzed, but such relationships showed better agreement for the more dominant taxonomic groups; and (3) ammonium hydroxide in ethanol had no effect on molecular diversity. Together, these results indicate that with appropriate replication, paired metabarcoding and morphological analyses can characterize zooplankton community structure and biomass, and that metabarcoding methods are to some extent indicative of relative community composition when absolute measures of abundance or biomass are not available.