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Leptospirosis is a significant zoonotic disease affecting livestock, leading to reproductive issues and economic losses. Despite its endemic status in India, research has predominantly focused on coastal regions, leaving the North Eastern Region (NER) underexplored. This study aims to investigate the seroprevalence and serogroup distribution of leptospirosis in livestock across Assam, a major state in the North Eastern Region (NER) of India. Serum samples (n=811) from cattle, buffalo, sheep, goats, and pigs were collected between 2016 and 2019 and screened using the Microscopic Agglutination Test (MAT) for 24 serogroups. The overall seroprevalence was 22.9 % (186/811), with highest prevalence in cattle (26.2 %) and buffalo (25 %), followed by small ruminants (19.8 %) and pigs (18.6 %). Notably, uncommon serovars such as Mini (28.8 %), Manhao (12.4 %), and Cynopteri (7.5 %) were identified, indicating a unique epidemiological pattern in Assam. High seroprevalence was observed in districts like Bongaigaon (66.7 %), Kamrup Metropolitan (50.0 %), and Nalbari (40.0 %), emphasizing the need for targeted intervention strategies. The presence of these uncommon serogroups, typically found in neighbouring countries and other regions, suggests potential transboundary transmission from these countries. This study provides valuable insights into the seroprevalence and serogroup distribution of leptospirosis in Assam's livestock, highlighting the need for region-specific surveillance and control measures. These findings underscore the importance of understanding the local epidemiological landscape to develop effective disease management and prevention strategies, ultimately reducing the impact of leptospirosis in the NER of India. • This study reports a 22.9 % seroprevalence of leptospirosis in livestock across Assam indicating public health concerns. • It is the first to test 24 Leptospira serogroup in Assam, revealing a diverse range of serovar prevalence in livestock. • Cattle (26.2%) and buffalo (25%) exhibited the highest seroprevalence rates among livestock. • High-prevalence districts: Bongaigaon (66.7 %), Kamrup Metropolitan (50.0 %), & Nalbari (40.0 %) need targeted interventions. • The discovery of serovars Mini, Manhao, and Cynopteri in Assam's livestock differs from patterns observed in rest of India. • This suggests potential transboundary transmission & indicates the need of region-specific surveillance and control measures. [ABSTRACT FROM AUTHOR]