Human astrovirus (HAstV) is an important pathogen that causes acute gastroenteritis mainly in children up to 5 years old worldwide. A total of 318 fecal samples were collected from January to December of 2004 from children with acute gastroenteritis hospitalized in Rio de Janeiro (RJ), Brazil. The samples were obtained from three public hospitals and were previously investigated for the presence of rotavirus (RV) and enteric adenovirus (AdV). The aim of this study was to determine the prevalence and molecular characterization of HAstV by reverse-transcription polymerase chain reaction (RT-PCR) and partial sequencing of open reading frame 2 (ORF2). HAstV infections were detected in 43 (14%) of the samples, of which 19 (6%) were mixed infections with HAstV and RV. HAstV were identified in all age groups, with 85% of the cases occurring in children younger than 2 years old. HAstV infections were more prevalent from March to May. Diarrhea, vomiting, and fever were the main clinical manifestations observed in these infections. Partial capsid sequencing of 35 HAstV strains characterized 25 (71%) as genotype 1 (HAstV-1), 6 (17%) HAstV-2, 3 (9%) HAstV-8, and 1 (3%) HAstV-4. Based on the genetic variability, three lineages were observed between RJ HAstV-1 strains. This study demonstrated the importance of HAstV infections causing acute infantile gastroenteritis in hospitalized children in RJ, Brazil. Molecular epidemiological surveillance is helpful for designing prevention strategies of HAstV transmission.