Although　heterotrophic　nitrification　and　aerobic　denitrification　(HN-AD)　bacteria,　a　novel　functional　group　involved　in　nitrogen　conversion,　have　been　isolated　and　characterized,　the　lack　of　specific　molecular　markers　for　identification　severely　limits　the　study　of　their　role　in　geochemical　cycling　and　the　contribution　in　ecosystems.　Here,　a　set　of　molecular　markers　was　developed　for　the　rapid　identification　of　HN-AD　bacteria,　via　delving　into　the　genomics　and　transcriptomics　of　a　HN-AD　isolate　(Pseudomonas　aeruginosa　SNDPR-01).　Among　the　nine　candidate　genes　that　were　significantly　expressed　during　heterotrophic　nitrification,　three　were　involved　in　the　conversion　of　hydroxylamine　to　nitrite,　a　characteristic　process　of　HN-AD.　The　universality　and　stability　of　the　identification　methods　based　on　the　gene　primer　set　were　validated　using　pure　HN-AD　strains,　mixed　cultures　of　pure　HN-AD　strains,　and　activated　sludge　from　laboratory-scale　and　real　wastewater　treatment　plants.　In　all　cases,　the　amplification　outcome　was　positively　correlated　with　the　function　and　population　of　HN-AD　bacteria,　demonstrating　its　validity　as　a　molecular　marker.　This　study　supports　the　paradigm　of　heterotrophic　nitrification　from　hydroxylamine　to　nitrite.　As　an　effective　tool　for　the　identification　of　classic　HN-AD　bacteria,　this　study　lays　the　groundwork　for　research　on　environmental　ecology　and　biotechnological　application　of　HN-AD　bacteria.