The aim of this study was to investigate the presence of the recently identified human astrovirus (HAstV) and to increase the knowledge of the molecular epidemiology of classical HAstV detected in Uruguay. METHODS AND RESULTS: Recently identified and classical HAstV genotypes were investigated by RT-PCR targeting the ORF1b and ORF2 genome regions in 20 samples obtained between September 2011 and April 2013 in two cities of the eastern region of Uruguay. Four of 20 samples (20%) were identified as MLB-1 genotype and it was found a new MLB-1 classification through the segregation of the worldwide reported MLB-1 strains in two genetic lineages proposed and named: MLB-1a and MLB-1b. Fourteen (70%) samples were positive for classical HAstV and 12 of them were successfully sequenced and genotyped as: HAstV-1 (n = 10), HAstV-2 and HAstV-5 (one sample each). CONCLUSION: These results constitute the first report in the Latin American region concerning the molecular detection and characterization of MLB-1 HAstV strains in environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the benefits of an environmental surveillance to study emerging enteric viruses circulating in human societies.