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Lassa fever is a neglected disease in West Africa. The rodent vector, Mastomys natalensis and related species continue to flourish without strategic intervention in the associated ecological environment. The spatio-temporal pattern of Lassa fever (LF) cases among humans within an environment is essential for identifying the risk factors associated with the disease. This study examined the spatio-temporal pattern of Lassa fever cases and association with age, gender and seasonal frequency in Owo, Ondo State, Nigeria, during a three-year retrospective period (2018-2020). The study adopted a combination of hospital case review and a cross-sectional survey research design. A retrospective distribution analysis on reported cases of LF, mapped to show the distribution of human cases in Owo from January 2018 to December 2020, was carried out. Data on age, gender, date of illness, dry and wet season frequency were used to compute analysis of association between variables and LF pattern in Owo. The mean age of LF case was 55 years and standard deviation of 13 years. Two percent of the respondents were 26-30 years old, 7.9% were 31-35 years old, 30% were 36-40 years old and 60.2% were above 41 years old. Males (57.2%) accounted for a higher proportion of LF cases compared to the female (42.8%) counterparts. A total of 301 laboratory-confirmed cases were studied during the three-year retrospective period. Spatio-temporal pattern identified was categorized by clustered cases in three streets (p<0.01). A clear spread in Ehin-Ogbe Street, Aruwajoye Street and Oke-Dogbon Street was found. Subsequent sporadic spread into adjacent communities of clusters was found. LF confirmed cases were highest in the dry season as compared to the rainy season. Spatio-temporal clusters of the cases were highly significant (p -= 0.00048) between November and February corresponding to the dry season.These findings provide an empirical basis for priority areas for implementing preventive intervention to reduce LF burden in Owo.