This article evaluates the potential of 16S rRNA gene sequencing for species and strain-level microbiome analysis. It highlights that while short-read sequencing of 16S variable regions cannot achieve the taxonomic resolution afforded by sequencing the full 1500 bp gene, full-length sequencing platforms can accurately resolve subtle nucleotide substitutions but not insertions/deletions. The study argues that modern analysis approaches must account for intragenomic variation between 16S gene copies. Using in-silico and experimental data, it demonstrates that full-length 16S sequencing can provide species and strain-level taxonomic resolution. The study also shows that intragenomic 16S gene copy variants are prevalent in the human gut microbiome and can be used to improve discrimination between species and strains. The results indicate that full-length 16S sequencing provides better taxonomic resolution than partial sequencing, and that appropriate clustering of intragenomic variation can enable accurate classification of bacterial species. The study concludes that full-length 16S sequencing has the potential to enable accurate classification of individual organisms at very high taxonomic resolution.This article evaluates the potential of 16S rRNA gene sequencing for species and strain-level microbiome analysis. It highlights that while short-read sequencing of 16S variable regions cannot achieve the taxonomic resolution afforded by sequencing the full 1500 bp gene, full-length sequencing platforms can accurately resolve subtle nucleotide substitutions but not insertions/deletions. The study argues that modern analysis approaches must account for intragenomic variation between 16S gene copies. Using in-silico and experimental data, it demonstrates that full-length 16S sequencing can provide species and strain-level taxonomic resolution. The study also shows that intragenomic 16S gene copy variants are prevalent in the human gut microbiome and can be used to improve discrimination between species and strains. The results indicate that full-length 16S sequencing provides better taxonomic resolution than partial sequencing, and that appropriate clustering of intragenomic variation can enable accurate classification of bacterial species. The study concludes that full-length 16S sequencing has the potential to enable accurate classification of individual organisms at very high taxonomic resolution.