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Anticoagulants are a major component of rodenticides used worldwide, which function by effectively blocking the vitamin K cycle in rodents. The rat Vitamin K epoxide Reductase Complex (VKORC) subunit 1 is the enzyme responsible for recycling vitamin K, and five substitution mutations (Tyr139Cys, Tyr139Ser, Tyr139Phe and Leu128Gln and Leu120Gln) located in the VKORC1 could result in resistance to anticoagulant rodenticides. This study carried out a VKORC1-based survey to estimate the anticoagulant rodenticide resistance in three Rattus species (R. losea, R. norvegicus, and R. tanezumi) collected in Hong Kong. A total of 202 rats captured in Hong Kong between 2017 and 2021 were analysed. Sequencing of molecular marker cytochrome c oxidase subunit 1 (COX1) was carried out to assist the species identification, and the identities of 52 lesser ricefield rats (R. losea), 81 common rats (R. norvegicus) and 69 house rats (R. tanezumi) were confirmed. Three VKORC1 exons were amplified from individuals by PCR followed by Sanger sequencing. A total of 47 R. tanezumi (68.1%) contained Tyr139Cys mutation in VKORC1 gene, and half of them were homozygous. None of the collected R. losea and R. norvegicus were detected with the five known substitutions leading to anticoagulant rodenticides resistance, and previously undescribed missense mutations were revealed in each species. Whole genome sequencing was further carried out on some individuals, and single nucleotide polymorphisms (SNPs) were also identified in the introns. This is the first study investigating the situation of anticoagulant rodenticide resistance in the rats collected in Hong Kong. Given that the efficacy of rodenticides is crucial for effective rodent management, regular genetic testing as well as population genomic analyses will be required to both monitor the situation and understand the adaption of different rat haplotypes for integrated pest management. Susceptibility tests for individual rodenticides should also be conducted regularly to assess their effectiveness on local species.