We have recently developed a method that can produce millions of high-quality, full-length 16S rRNA sequences from any environment. Here we used this method to generate 412,174 of such sequences from anaerobic digesters located at Danish wastewater treatment plants. The sequences were used to create the first comprehensive anaerobic digester specific 16S rRNA reference database containing 9,174 unique full-length 16S rRNA sequences. Phylogenetic analyses of the database revealed many novel microbes, which highlights large gaps in our current knowledge regarding the microorganism involved in the anaerobic digestion process. The reference database now allows us to assign provisional names to these organisms and start to connect them with their ecological roles in the AD process.