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Rodent-borne hantaviruses are pathogenic in humans and remain a threat for public health. In Asia, data are available mostly from oriental countries, but limited in South-East Asia. In Cambodia, the presence of hantavirus was reported in rodents since 1998. Nevertheless, burden of infection in humans remains unknown. In our study, we aimed to document the presence and diversity of hantaviruses in rodents and to assess hantavirus seroprevalence in people living in urban and rural settings in Cambodia. This is a retrospective study conducted using pooled organs collected from 750 rodents in urban and rural settings in 2020 (rainy season) and 2022 (dry season). Concurrently, 788 people working or living at the vicinity of rodent trapping locations were requested for blood collection. Hantavirus RNA in rodents was assessed using RT-PCR and confirmed by Sanger sequencing. Human sera were tested for anti-Thailand hantavirus IgG and IgM using in-house ELISA. In rodents, hantavirus RNA was detected in 25 (3.3%) caught rodents all originated from urban area. Seoul orthohantavirus was the most predominant virus detected in 23 Rattus norvegicus and 1 Rattus rattus. Thottapalayam virus was detected in one Suncus murinus. In humans, the initial seroprevalence of anti-hantavirus IgG was 10.0% (79/788) in 2020. For 70.4% (555/788) of these study participants, a follow-up blood collection was performed in 2022. Out of these, six (1.1%) individuals turned seroconverted, and one even was positive for hantavirus IgM antibodies. Overall, the hantavirus seroprevalence was significantly higher in urban compared to rural area (13.8% vs. 8.9%, p=0.03). Our findings highlighted a new insight of hantavirus circulation at the rodent-human interface in Cambodia. Additional analyses are ongoing to identify potential factors associated with human exposure to hantavirus. Further investigations are also needed to determine the impact of hantavirus infection on human health in Cambodia. [ABSTRACT FROM AUTHOR]