Aims: To assess human adenovirus (HAdV) diversity in environmental samples based on sequence comparisons of hexon gene fragments amplified using newly designed HAdV-specific polymerase chain reaction (PCR) assays. Methods and Results: Six PCR primer sets were designed based on 56 aligned hexon sequences from NCBI GenBank to amplify different hexon gene sections (241-349 bp) of the six HAdV species. The amplified hexon genes from wastewater samples were cloned, sequenced, and compared with those in publicly accessible databases (i.e. NCBI GenBank) by using the Blast program. A total of 46 analysed positive clones were affiliated to five HAdV serotypes, i.e. 1, 2, 12, 31 and 41. Similarities between the cloned and database hexon sequences ranged from 95 center dot 9 to 100% (with an average of 98 center dot 1 +/- 1 center dot 0%). Conclusion: The designed primers showed higher amplification efficiencies when compared with the existing assays. Using the new assays, HAdV species A, C, and F (serotypes 1, 2, 12, 31 and 41 in particular) were identified in the studied municipal wastewater. Significance and Impact of the Study: The six PCR primer sets developed in this study can be used to efficiently amplify hexon gene fragments in HAdV. Multiple HAdV serotypes identified in the municipal wastewater provide new information about HAdV diversity in environmental samples.