Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the biogeochemical cycling of nitrogen. They derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. Several methods have been used to detect the presence and activity of anammox bacteria in the environment, including 16S rRNA gene-based approaches. The use of the 16S rRNA gene to study biodiversity has the disadvantage that it is not directly related to the physiology of the target organism and that current primers do not completely capture the anammox diversity. Here we report the development of PCR primer sets targeting a subunit of the hydrazine synthase (hzsA), which represents a unique phylogenetic marker for anammox bacteria. The tested primers were able to retrieve hzsA gene sequences from anammox enrichment cultures, full-scale anammox wastewater treatment systems, and a variety of freshwater and marine environmental samples, covering all known anammox genera.