Multiple subtypes of influenza A viruses circulating in animals must be closely monitored to understand their risk to humans and animal populations. Many molecular-based subtyping methods require constant monitoring of viral genomes for primer and/or probe mismatches and are prone to primer-primer interactions. This report presents a new approach that involves target enrichment through cDNA hybridization followed by adapter-mediated amplification for subtyping influenza virus (AmASIV). As a proof of concept, the AmASIV assay was multiplexed to specifically detect and differentiate influenza A virus subtypes (H5, N5, N7, and N9) in a single reaction without cross-recognition of nontarget subtypes or influenza B virus. The limit of detection (LOD) of AmASIV, as measured by 50 % egg-infective dose per reaction (EID50/reaction), was comparable to that of singleplex TaqMan (R) qPCR assays with LODs of 10(-0.6) (H5), 10(2) (N5), 10(-0.3) (N7), and 10(-0.5) (N9) EID50/reaction. The AmASIV will strengthen animal influenza virus surveillance and laboratory capacity to improve prevention and control of influenza.