This protocol describes the preparation of Illumina libraries for high through-put sequencing of near full-length\u00a0bacterial 16S rRNA sequences. The method is based on the universal 27F and 1492R primers designed by Lane et al.*Sequences for primers and adaptor\u00a0used can be found under guidelines.*Lane, D. J. (1991). 16S\/23S rRNA sequencing. In E. Stackebrandt & M. Goodfellow (Eds.), Nucleic Acid Techniques in Bacterial Systematics (pp. 115\u2013175). Chichester, United Kingdom: John Wiley and Sons.