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Background: In this study, we carried out an investigation of Kyasanur Forest Disease (KFD) suspected human cases reported in Karnataka state, India from December 2018 to June 2019., Methods: The clinical samples of KFD suspected cases ( n  = 1955) from 14 districts of Karnataka were tested for KFD using real-time RT-PCR and IgM ELISA. Further, the KFD-negative samples were tested for IgM antibodies against dengue and chikungunya viruses. Monkey samples ( n  = 276) and tick pools ( n  = 11582) were also screened using real-time RT-PCR. KFD-positive samples were further analysed using next-generation sequencing along with clinico-epidemiological analysis., Results: Of all, 173 (8.8%) cases tested positive for KFD either by real-time RT-PCR ( n  = 124), IgM ELISA ( n  = 53) or both tests ( n  = 4) from seven districts. Among KFD-negative cases, IgM antibody positivity was observed for dengue (2.6%), chikungunya (5.8%), dengue and chikungunya coinfection (3.7%). KFD cases peaked in January 2019 with fever, conjunctivitis, and myalgia as the predominant symptoms and a mortality of 4.6%. Among confirmed cases, 41% received a single dose and 20% received two doses of the KFD vaccine. Of the seven districts with KFDV positivity, Shivamogga and Hassan districts reported KFD viral RNA positivity in humans, monkeys, and ticks. Sequencing analysis of 2019 cases demonstrated a difference of less than 1.5% amino acid compared to prototype KFDV., Conclusion: Although the KFD has been endemic in many districts of Karnataka state, our study confirms the presence of KFDV for the first time in two new districts, i.e. Hassan and Mysore. A comparative analysis of KFDV infection among the KFD-vaccinated and non-vaccinated populations demonstrated an insignificant difference.