Acute infectious diarrhea (AcID) causes a significant health burden on the population of developed countries, and even a higher burden in the developing world. The only investigation into the causes of AcID in Winnipeg was conducted in the late 1970s. That study determined that 3%-5% of Children’s Hospital Emergency Room (CHER) visits were due to AcID, and about 50% of those were due to rotavirus infections. We conducted a prospective case-control study to reveal the current spectrum of viral pathogens associated with AcID and the distribution and frequency of their occurrence among the pediatric population of Winnipeg. In this study, nucleic acid detection (NAD) and genome sequence information confirmed the presence and identity of each pathogen, and established whether an etiological shift in the distribution of pathogens, both between families and strains of specific pathogens, occurred. Stool samples were collected from pediatric cases with AcID at the ER along with asymptomatic cases for control. A panel of viral nucleic acid detection (NAD) assays was established by the Viral Gastroenteritis Study Group for human astro, calici (Noro and Sapo), entro, polio, hepA, rota and reo viruses according to the published procedures. A new assay for Aichivirus was developed, and the VGSG has established a novel rotavirus assay which is capable of detecting rotavirus from at least 4 different host species (Human, bovine, porcine and simian). Amplified viral targets were sequenced and the information submitted to GeneBank to confirm the strain of each isolate. A total of 1128 patients visited WCHER and WC during the study period and among them 242 patients were enrolled. In 104 cases viruses were identified. A total number of 114 viruses were identified either by NAD or EM assay. Out of 114 viruses, prevalence of HAdV, NoV GI/II and HRV were 44%, 23% and 23% respectively. Mixed infections were found in 4% of cases.This knowledge of pathogen distribution will facilitate design of effective methods for prevention, treatment and intervention in the spread of AcID pathogens.