Abstract The 16S ribosomal RNA (rRNA) gene is widely used to survey microbial communities. Sequences are often clustered into Operational Taxonomic Units (OTUs) as proxies for species. The canonical clustering threshold is 97% identity, which was proposed in 1994 when few 16S rRNA sequences were available, motivating a reassessment on current data. Using a large set of high-quality 16S rRNA sequences from finished genomes, I assessed the correspondence of OTUs to species for five representative clustering algorithms using four accuracy metrics. All algorithms had comparable accuracy when tuned to a given metric. Optimal identity thresholds that best approximated species were ∼99% for full-length sequences and ∼100% for the V4 hypervariable region.