Anammox (anaerobic ammonium oxidation) is a microbial-mediated pathway that plays an essential role in nitrogen cycling. Over the past twenty years, the characteristics of the anammox process and microbial metabolism have been determined by ecological, physiological, and genomic investigations. Anammox bacteria oxidize nitrite to nitric oxide via nitrite reductase, after which the nitric oxide further combines with ammonium to synthesize hydrazine, which is catalyzed by hydrazine oxidase to produce nitrogen gas. Functional genes encoding key enzymes in the anammox process have provided new molecular markers for ecological studies of anammox bacteria, and are potentially superior to 16S rRNA genes in linking anammox diversity to ecological functions. In this review, research on the key enzymes involved in the anammox process is introduced, and major advances in the molecular ecology of anammox bacteria are summarized based on functional gene investigations.