DNA metabarcoding studies of diverse assemblages like zooplankton typically target the conserved nuclear 18S rDNA, sacrificing taxonomic resolution to take advantage of its broad taxonomic coverage. Mitochondrial COI offers better taxonomic resolution and can take advantage of growing DNA barcode databases; however, several studies have demonstrated the taxonomic bias inherent in many COI markers due to the lack of conserved primer-binding sites. PCR-bias is also likely to reduce the utility of COI markers for estimating relative abundance from high-throughput sequencing (HTS) data. To avoid taxonomic bias but retain taxonomic resolution, mitochondrial 12S and 16S rDNA have been proposed as alternative metabarcoding markers. We used HTS to compare the performance of 18S, COI and 16S metabarcoding markers for characterizing zooplankton assemblages. In contrast to previous studies that recommend 18S, we find degenerate COI primers provide the best taxonomic coverage, taxonomic resolution and agreement between morphology- and DNA-based identifications. All markers revealed similar patterns of beta-diversity, although different taxa were identified as the greatest contributors to these patterns for 18S compared to the mitochondrial markers and the morphology-based analysis. Interestingly, all markers showed potential for retrieving semi-quantitative abundance data; however, 18S provided the strongest relationship between biomass and number of HTS reads. Our results show the taxonomic coverage and resolution provided by degenerate COI primers, combined with a comparatively well-developed reference sequence database, make them valuable metabarcoding markers for biodiversity assessment.