Due to the high divergence among 16S rRNA genes of anammox bacteria, different diversity pattern of the community could be resulted from using different primer set. In this study, the efficiencies and specificities of two commonly used sets, Amx368F/Amx820R and Brod541F/Amx820R, were evaluated by exploring the diversity characteristics of anammox bacteria in sediments from marine, estuary, and freshwater wetland. Statistical analysis indicated that the base mispairing rate between bases on 16S rRNA gene sequences retrieved by Amx368F/Amx820R and their corresponding ones on primer Brod541F was quite high, suggesting the different efficiency and specificity of Amx368F/Amx820R and Brod541F/Amx820R. Further experimental results demonstrated that multiple genera of anammox bacteria, including Ca. Scalindua, Ca. Brocadia, and Ca. Kuenenia, were able to be detected by Amx368F/Amx820R, but only Ca. Scalindua could be retrieved by Brod541F/Amx820R. Moreover, the phylogenetic clusters of Ca. Scalindua by Amx368F/Amx820R were different completely from those by Brod541F/Amx820R, presenting a significant complementary effect. By joint application of these two primer sets, the diversity distribution patterns of anammox bacteria in different environments were analyzed. Almost all retrieved sequences from marine sediments belonged to Ca. Scalindua. Sequences from freshwater wetland were affiliated to Ca. Brocadia and two new clusters, while high diversity of anammox bacteria was found in estuary, including Ca. Scalindua, Ca. Brocadia, and Ca. Kuenenia, corresponding to the river-sea intersection environmental feature. In total, these two prime sets have different characteristic for anammox bacteria detecting from environmental samples, and their combined application could achieve better diversity display of anammox community.