The real-time polymerase chain reaction was used to quantitatively evaluate distribution patterns and nitrogen removal pathways of the amoA, nxrA, narG, napA, nirK, qnorB, nosZ, nas, and nifH genes and 16S rRNA in anaerobic ammonia oxidation bacteria in four multimedia constructed wetlands for rural wastewater treatment. The results indicated that the abundance of functional genes for nitrogen removal in the rhizosphere layer (0 to 30 cm), water distribution layer (30 to 50 cm), multime filler layer (50 to 130 cm), and catchment layer (130 to 170 cm) of the constructed wetlands were closely related. The rhizosphere layer was conducive to the absolute enrichment of dominant genes. The other three layers were favorable to the relative enrichment of rare genes.