The prevalence and seasonal variation of 7 viruses in 6 major rivers in Alberta were assessed using a combination of qPCR, cell culture and integrated cell culture with qPCR (ICC-qPCR). Water samples were collected monthly from rivers at different sites upstream and downstream of major urban centers. Seven viruses including rotavirus, adenovirus, astrovirus, norovirus, sapovirus, JC virus and enterovirus, were detected in at least one of the water samples at each site using qPCR. Rotavirus was most common with concentration ranging from 2.3 to 4.5 log10 genomic equivalent (GE) copies/L. Norovirus, sapovirus, astrovirus, adenoviruses and JC virus peaked during the winter (November to March). Viruses were most prevalent at the Bow River sampling site downstream of the City of Calgary, followed by the North Saskatchewan River site downstream of the City of Edmonton and the Red Deer River site downstream of the City of Red Deer. The detection rates and quantity of viruses had significant difference in the sampling sites between upstream and downstream of major urban centers (p < 0.001). 14% of the samples tested positive using viral culture indicating the presence of infectious viruses in river. Sequencing analysis identified human rotavirus in 75% of the samples collected from downstream versus 37% of the samples collected from upstream sites (p < 0.02). Multivariate binary regression showed that human activity in watersheds is a significant determinant of viruses in Alberta's Rivers. The discharge from wastewater treatment plants may be the possible sources of viral contamination. Seasonal coincidence of acute viral gastroenteritis outbreaks and monthly peak occurrence of enteric viruses in river water implies potential impact of waterborne viruses on human health. Display omitted •Seven viruses were detected in 6 major rivers across Alberta by qPCR.•Rotavirus and adenovirus were the most common viruses detected all year around.•Norovirus, sapovirus, astrovirus and adenovirus had seasonal peak during winter.•Viruses were more frequently detected at downstream of cities along the river.•Infectious viruses were detected in 14% of the samples by cell culture and ICC-qPCR.