Rotavirus is the most common cause of severe infectious diarrhea worldwide in children under 5 years of age. We have evaluated the performance of multiplex PCR [Seeplex® Diarrhea-V ACE Detection (V2.0) Kit] (the version 2.0 kit) for detecting rotaviruses in human stool specimens, in a comparison with ELISA (RIDASCREEN®) and the older Seeplex kit, namely Seeplex® Diarrhea-V ACE Detection Kit (the original kit). A total of 173 stool specimens, previously tested for rotavirus infection by ELISA from May 2014 to March 2016, were subjected to the two Seeplex® kits. The positive rate for the detection of rotavirus was 46.2% (80/173) by the version 2.0 kit. As compared with ELISA, the agreement rate of the original kit and the version 2.0 kit was 64.2% (positive agreement rate 23.1%, negative agreement rate 97.9%) and 97.7% (positive agreement rate 98.7%, negative agreement rate 96.8%), respectively. The agreement rate between the original kit and the version 2.0 kit was 65.3%. Thus, the Seeplex® Diarrhea-V ACE Detection (V2.0) Kit showed acceptable clinical performance and could be useful for detecting rotavirus infection with a performance superior to the original kit using the ELISA as the comparator.