Epidemiological data from Latin America on acute gastroenteritis (AGE) in the post rotavirus vaccine era obtained using highly sensitive molecular techniques are scarce.This prospective surveillance study was performed between March 15, 2015 and March 19, 2016 in two municipal health networks (MHNs) in Cali, Colombia to detect AGE in children <5 years of age. Consecutive sampling was performed simultaneously in all health facilities belonging to both MHNs until completion of the required sample size. Stool samples from AGE patients were tested with a nucleic acid assay for 16 pathogens. Detection frequency and incidence rates were obtained for specific pathogens according to age-group in children with AGE leading to hospitalization or outpatient care.Overall incidence rates of AGE-related hospitalization and outpatient care were 20 and 237 per 1000 children <5 years of age, respectively. Despite almost complete rotavirus vaccine uptake, rotavirus was the most common etiology overall, including hospitalization and outpatient treatment of 0-23-month-olds, with incidence rates of 12 and 108 per 1000 children, respectively. Norovirus incidence rates were similar to rotavirus rates in this age group and associated with high Vesikari scores. Shigella predominated in 24-59-month-olds.AGE remains an important cause of morbidity in children under 5 years of age, especially in those under 2 years. Rotavirus remains the leading AGE-associated pathogen, followed closely by norovirus in younger children. Preventive measures, including novel vaccination strategies, are necessary in this population to further reduce AGE-related morbidity.