The major sites of water column denitrification in the ocean are oxygen minimum zones (OMZ), such as one in the eastern South Pacific (ESP). To understand the structure of denitrifying communities in the OMZ off Chile, denitrifier communities at two sites in the Chilean OMZ (Antofagasta and Iquique) and at different water depths were explored by terminal restriction fragment length polymorphism analysis and cloning of polymerase chain reaction (PCR)-amplified nirS genes. NirS is a functional marker gene for denitrification encoding cytochrome cd1-containing nitrite reductase, which catalyses the reduction of nitrite to nitric oxide, the key step in denitrification. Major differences were found between communities from the two geographic locations. Shifts in community structure occurred along a biogeochemical gradient at Antofagasta. Canonical correspondence analysis indicated that O2, NO3–, NO2– and depth were important environmental factors governing these communities along the biogeochemical gradient in the water column. Phylogenetic analysis grouped the majority of clones from the ESP in distinct clusters of genes from presumably novel and yet uncultivated denitrifers. These nirS clusters were distantly related to those found in the water column of the Arabian Sea but the phylogenetic distance was even higher compared with environmental sequences from marine sediments or any other habitat. This finding suggests similar environmental conditions trigger the development of denitrifiers with related nirS genotypes despite large geographic distances.