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Abstract Leptospirosis is a ubiquitous disease and a major clinical challenge owing to the multitude of clinical presentations and manifestations that are possibly attributable to the diversity of Leptospira , the understanding of which is key to study the epidemiology of this emerging global disease threat. Sri Lanka is a hotspot for leptospirosis with high levels of endemic disease as well as annual epidemics. We carried out a prospective study of Leptospira diversity in Sri Lanka, covering the full range of climatic zones, geography, and clinical severity. Samples were collected for leptospiral culture from 1192 patients from 15 of 25 districts in Sri Lanka over two and half years period. Twenty-five isolates belonging to four pathogenic Leptospira species were identified: L. interrogans , L. borgpetersenii , L. weilii , and L. kirschneri . At least six serogroups were identified among the isolates: Autumnalis (6), Pyrogenes (4), Icterohaemorrhagiae (2), Celledoni (1), Grippotyphosa (2) and Bataviae (1). Seven isolates did not agglutinate using available antisera panels, suggesting new serogroups. Isolates were sequenced by Illumina. These data add 25 new core genome sequence types and were clustered in 15 clonal groups, including 12 new clonal groups. L. borgpetersenii was found only in the dry zone and L. weilii only in the wet zone. Acute kidney injury and cardiovascular involvement were seen only with L. interrogans infections. Thrombocytopenia and liver impairment were seen in both L. interrogans and L. borgpetersenii infections. The inadequate sensitivity of culture isolation to identify infecting Leptospira species underscores the need for culture-independent typing methods for Leptospira . Author Summary There is a huge diversity in pathogenic Leptospira species worldwide, and our knowledge of the currently circulating species is deficient owing to limited isolation and identification of Leptospira species from endemic countries. This prospective study reveals the wide pathogen diversity that causes human leptospirosis in Sri Lanka, representing four species, more than eight serogroups, and fifteen clonal groups. Further, the different geographic and climatic zone distributions and clinical manifestations observed underscores the need for prospective studies to expand the molecular epidemiological approaches to combat leptospirosis