Previously published NA subtype-specific real-time reverse-transcriptase PCRs (RRT-PCRs) were further validated for the detection of five avian influenza virus (AIV) NA subtypes, namely N5, N6, N7, N8, and N9. Testing of 30 AIV isolates of all nine NA subtypes informed the assay assessments, with the N5 and N9 RRT-PCRs retained as the original published assays while the N7 and N8 assays were modified in the primer–probe sequences to optimize detection of current threats. The preferred N6 RRT-PCR was either the original or the modified variant, depending on the specific H5N6 lineage. Clinical specimen (n = 137) testing revealed the ability of selected N5, N6, and N8 RRT-PCRs to sensitively detect clade 2.3.4.4b highly pathogenic AIV (HPAIV) infections due to H5N5, H5N6, and H5N8 subtypes, respectively, all originating from European poultry and wild bird cases during 2016–2018. Similar testing (n = 32 clinical specimens) also showed the ability of N7 and N9 RRT-PCRs to sensitively detect European H7N7 HPAIV and China-origin H7N9 low pathogenicity AIV infections, respectively.