In this study, the seasonal difference and the observable presence/absence of human adenovirus (HAdV) in the Puzih River basin in Taiwan was investigated. A total of 288 water samples were collected from 24 sites from March 2014 to February 2015. Human AdV analysis of sample was subjected to viral concentration using a GN-6 Metricel® filter, followed by DNA extraction, nested-PCR, and qPCR. Human AdV was detected in 34.3 % (99/288) of the entire river water sample. A higher percentage of HAdV (76.4 %) was obtained during the winter. The HAdV median concentration was relatively high in fall (1.4 × 10(3) copies/L) and winter (2.8 × 10(3) copies/L). Significant difference and correlation were found between the seasonal variation of HAdV and water quality parameters, including heterotrophic plate count, total coliform, water temperature, and turbidity. The most frequently identified HAdV (subgenus F) serotype was 41. Human AdV-41 is the main cause of gastroenteritis and should be considered for associated human health risk potential in the Puzih River basin.