This prospective study was performed to evaluate and compare the performance of the multiplex PCR Seeplex® assays and Anyplex™ II assays. From May 2014 until April 2016, a total of 247 respiratory samples were collected in Okinawa, Japan. Multiple respiratory pathogens were detected in 37% of patients with positive results. The most prevalent pathogens were influenza A virus and respiratory syncytial virus B. Despite minor differences in capabilities, both the Seeplex® assays and Anyplex™ II assays can be easily implemented in diagnostic or research laboratories to optimize the detection and management of respiratory pathogen induced diseases.