Mixed community or environmental DNA marker gene sequencing has become a commonly used technique for biodiversity analyses in freshwater systems. Many cytochrome c oxidase subunit I (COI) primer sets are now available for such work. The purpose of this study is to test whether COI primer choice affects the recovery of arthropod richness, beta diversity, and recovery of target assemblages in the benthos kick-net samples typically used in freshwater biomonitoring. We examine six commonly used COI primer sets on samples collected from six freshwater sites. Biodiversity analyses show that richness is sensitive to primer choice and the combined use of multiple COI amplicons recovers higher richness. Thus, to recover maximum richness, multiple primer sets should be used with COI metabarcoding. In ordination analyses based on community dissimilarity, samples consistently cluster by site regardless of amplicon choice or PCR replicate. Thus, for broadscale community analyses, overall beta diversity patterns are robust to COI marker choice. Recovery of traditional freshwater bioindicator assemblages such as Ephemeroptera, Trichoptera, Plectoptera, and Chironomidae as well as Arthropoda site indicators were differentially detected by each amplicon tested. This work will help future biodiversity and biomonitoring studies develop not just standardized, but optimized workflows that either maximize taxon-detection or the selection of amplicons for water quality or Arthropoda site indicators.