A comparative analysis was performed with 25 isolates of astroviruses (AstVs) detected in sewage sources and 22 concurrently identified clinical AstV isolates from the Tshwane (Pretoria) Metropolitan Area in South Africa. The samples and specimens were screened for AstVs by using an enzyme immunoassay and/or a reverse transcriptase PCR (RT-PCR) for the highly conserved untranslated region (3′ end) of the genome. The RT-PCR results were confirmed by oligonucleotide probe dot blot hybridization. Viable viruses were propagated in cell cultures for amplification when a minimal specimen was available or indeterminate sequences were obtained. AstV strains were characterized by RT-PCR and partial sequence analysis of the capsid region. The presence of multiple human AstV (HAstV) types in a single sewage sample complicated identification of individual strains, and additional type-specific RT-PCR and sequence analyses of the capsid region were required for characterization. Amplification and characterization of one genotype from a sample, therefore, did not preclude the possibility that a sample harbored additional different genotypes. Genotype and sequence information obtained from AstVs in wastewater samples were compared to information obtained from AstV strains from human stools. HAstV type 1 (HAstV-1), as well as HAstV-3, -5, -6, and -8, were identified among the clinical isolates, and HAstV-1, -2, -3, -4, -5, -7, and -8 were identified among the environmental samples. Phylogenetic analysis demonstrated that HAstV-1, -3, -5, and -8, which were present in human stool and sewage samples, clustered together, indicating that these viruses are closely related. The concurrent presence of identical HAstV strains in wastewater samples and in hospitalized patients suggests that AstVs present in the environment pose a potential risk to communities in which fecally contaminated water is used for recreational and domestic purposes.