Serious concern about the worldwide transmission of the Asian H5N1 highly pathogenic (HP) avian influenza (AI) virus by migratory birds surrounds the importance of the AI global surveillance in wild aquatic birds and underscores the requirement for a reliable subtyping method of AI viruses. PCR is advantageous due to its simplicity, less cross-reactivity and unlimited reagent supply. Currently, the only available HA subtyping primer set that can subtype H1 through H15 is not fully evaluated and, since it only targets HA1, is unavailable for molecular pathotyping of AI viruses. Our preliminary experiments found that these primer sets were cross-reactive and missed some recent AI viruses. In this study, we developed new primer sets against HA cleavage sites for subtyping of H1 to H15 genes and for molecular pathotyping. Our primer sets were subtype-specific and detected 99% of previously identified HA genes (115/116, 1949 – March 2006), and the correct amplification of HA genes were confirmed by sequence analyses of all 115 PCR products. The primer sets successfully subtyped most of the recent AI viruses isolated in Japan (96%, 101/105, October 2006 – March 2007). Taken together, our primer sets could efficiently detect HA genes (98%, 216/221) of both previous and recent HA genes or of both American (29/29) and Eurasian (187/192) lineages. All 38 H5 and 13 H7 viruses were molecularly pathotyped by sequencing analyses of the HA cleavage site. In contrast, despite efficient detection of previously identified strains (114/116, 98 %), the published primer sets exhibited lower specificity and lower detection efficiency against recent AI viruses (84/105, 80%). These results indicate that our primers are useful not only for HA subtyping but also for molecular pathotyping of both previous and recent AI viruses. These advancements will enable general diagnostic laboratories to subtype AI viruses for the surveillance in wild aquatic birds.