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Nitrification, the oxidation of ammonia via nitrite to nitrate, has always been considered to be catalyzed by the concerted activity of ammonia- and nitrite-oxidizing microorganisms. Only recently, complete ammonia oxidizers (“comammox”), which oxidize ammonia to nitrate on their own, were identified in the bacterial genus  Nitrospira , previously assumed to contain only canonical nitrite oxidizers.  Nitrospira  are widespread in nature, but for assessments of the distribution and functional importance of comammox  Nitrospira  in ecosystems, cultivation-independent tools to distinguish comammox from strictly nitrite-oxidizing  Nitrospira  are required. Here we developed new PCR primer sets that specifically target the  amoA  genes coding for subunit A of the distinct ammonia monooxygenase of comammox  Nitrospira . While existing primers capture only a fraction of the known comammox  amoA  diversity, the new primer sets cover as much as 95% of the comammox  amoA  clade A and 92% of the clade B sequences in a reference database containing 326 comammox  amoA  genes with sequence information at the primer binding sites. Application of the primers to 13 samples from engineered systems (a groundwater well, drinking water treatment and wastewater treatment plants) and other habitats (rice paddy and forest soils, rice rhizosphere, brackish lake sediment and freshwater biofilm) detected comammox  Nitrospira  in all samples and revealed a considerable diversity of comammox in most habitats. Excellent primer specificity for comammox  amoA  was achieved by avoiding the use of highly degenerate primer preparations and by using equimolar mixtures of oligonucleotides that match existing comammox  amoA  genes. Quantitative PCR with these equimolar primer mixtures was highly sensitive and specific, and enabled the efficient quantification of clade A and clade B comammox  amoA  gene copy numbers in environmental samples. The measured relative abundances of comammox  Nitrospira , compared to canonical ammonia oxidizers, were highly variable across environments. The new comammox  amoA -targeted primers enable more encompassing future studies of nitrifying microorganisms in diverse habitats. For example, they may be used to monitor the population dynamics of uncultured comammox organisms under changing environmental conditions and in response to altered treatments in engineered and agricultural ecosystems.

AmoA-Targeted Polymerase Chain Reaction Primers for the Specific Detection and Quantification of Comammox Nitrospira in the Environment