Aims: Outbreaks of hepatitis A in Thailand have been reported continuely and associated with water supply. However, the genetic analysis of hepatitis A virus (HAV) in water is limited. This study described the application of virus concentration method and reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) to detect HAV RNA and analyse the genetic sequence of the virus in environmental water samples. Methods and Results: The HAV from water samples was concentrated by using a developed virus concentration method (adsorption-elution and subsequent speedVac reconcentration) and the viral RNA was detected by RT-nested PCR followed by sequencing of the amplified DNA products. Detection limit of HAV determined by the RT-nested PCR was 1.29 radioimmunofocus assay (RIFA) units ml(-1). The DNA band appeared at 183 basepairs. No cross-reactivity was observed in the presence of other enteric viruses (poliovirus and rotavirus). A total of 180 water samples were collected, concentrated, and detected for HAV. The HAV was found in 6/40 (15%) of water samples collected from a swamp and 3/30 (10%) collected from a canal. Ten river samples and 100 tap water samples stored in containers for drinking and domestic uses were negative for HAV. In sequence analysis of the DNA products and alignment with the HAV sequence deposited in the GenBank, six water samples showed the nucleotide sequence associated with HAV. The 120 nucleotides in the N-terminal VP1 region obtained from two swamp samples showed 95 and 96.7% identity to HAV genotype IA. In nearly all water samples where HAV was present bacterial indicators (faecal coliforms and Escherichia coli) were found for faecal contamination. Conclusions: A coupled virus concentration method and RT-nested PCR was successfully applied to examine HAV in water samples collected from various sources. DNA sequencing of nested PCR products showed the genotype IA associated with HAV that is predominate in Thailand. Significance and Impact of the Study: This research is the first study of genetic sequence of HAV in water samples in Thailand. The presence of naturally occurring HAV might pose a potential health risk for people.