Anaerobic　ammonium　oxidizing　(anammox)　bacteria　are　widely　distributed　and　contribute　significantly　to　the　global　nitrogen　cycle.　Traditionally,　identification　and　quantification　based　on　the　16S　rRNA　gene　were　considered　not　reliable　because　of　low　16S　rRNA　gene　sequence　identity　within　Brocadiales.　Here　we　hypothesize　that　by　using　appropriate　primers　and　methodology,　16S-based　detection　and　quantification　of　anammox　bacteria　can　be　accurate.　We　modified　an　existing　16S　rRNA　gene-based　primer　pair　(Amx694F-Amx960R)　by　changing　one　nucleotide　(Amx694F　position　18,　G　-＞　C)　(Amx694PF-Amx960R)　so　that　they　match　the　sequences　of　most　Brocadiales　anammox　bacteria,　and　evaluated　the　modified　primer　pair　with　29　freshwater　samples　from　microcosms,　anammox　reactors　and　wastewater　treatment　plants　of　various　geographical　origins.　The　primer　pair　showed　high　specificity　in　detection　and　quantification　of　anammox　populations　in　samples　that　contained　＞0.1%　anammox　bacteria.　Quantification　of　anammox　abundance　by　quantitative　real-time　PCR　and　delineation　of　anammox　species　by　denaturing　gradient　gel　electrophoresis　agreed　well　with　amplicon　sequencing　results.　A　clear　shift　of　anammox　population　towards　＇Candidatus　Kuenenia＇　was　observed　under　laboratory　cultivation　conditions.　With　the　help　of　amplicon　sequencing,　we　demonstrated　that　16S　rRNA　gene-based　anammox-specific　primers　are　able　to　achieve　qualitative　and　quantitative　monitoring　of　anammox　communities　in　wastewater　treatment　plants　and　natural　freshwater　environments.2007;73:5261-7.