A study was conducted in agricultural and urban areas in Cambodia to assess the presence of hantaviruses in rodent populations. In 1998, rodents were trapped in two villages and in Phnom Penh city around market places and a rubbish dump. IgG antibodies to Hantaan virus were detected in 54 (8.2%) rodents among 660 tested: 6.4% (13/203) among roof rats (Rattus rattus), 20.9% (39/187) among Norway rats (R. norvegicus), 16.7% (2/12) among unidentified Rattus species and none in 183 Polynesian rats (R. exulans) or in 75 bandicoot rats (Bandicota sp.). The presence of the viral genome was detected by a reverse transcription-PCR amplifying part of the sequence coding for the nucleoprotein in the S segment, in 87% of the seropositive rodents. Thirty-one representative cDNAs were sequenced. Phylogenetic studies of the sequences indicated a close relationship with Seoul virus. However, the Cambodian Seoul virus sequences clustered within two different phylogenetic lineages, one associated with R. rattus and the other with R. norvegicus.