Objectives: We assessed the prevalence and genetic diversity of human adenovirus in children up to five years old, with and without acute gastroenteritis, in the Amazon region during 2016, 2017, and 2021. Design: This cross-sectional study used fecal samples from children admitted to a hospital in Boa Vista, Roraima. The samples were tested for adenovirus using quantitative real-time PCR and further characterized by PCR and sequencing of three specific genes. Results: Out of 942 samples, 13% tested positive for adenovirus—12% in children with acute gastroenteritis and 15.6% in children without gastroenteritis. Genotyping revealed that species F and C were predominant in acute gastroenteritis cases, while species F and A were more common in non-acute cases. The study found significant temporal variations in prevalence, with the highest rates in 2016. Adenovirus D37 was detected for the first time in Brazil. Conclusions: The study highlighted the notable presence and diversity of adenovirus among children in the Amazon region, emphasizing the need for continuous monitoring.