Leptospirosis is a globally important zoonotic disease caused by pathogenic Leptospira species, affecting humans and a broad range of domestic and wild animals. Despite its public health relevance, molecular evidence from the Himalayan region of India remains limited, particularly across multiple host groups within shared ecological settings. The present study aimed to investigate the occurrence and genetic identity of pathogenic Leptospira in humans and animals from the Darjeeling landscape of the Eastern Himalayas. A total of 294 samples, comprising human and domestic/peri-domestic animal specimens, were screened by LipL32-specific PCR for the detection of Leptospira. Overall, 15 samples (5.10%) tested positive, including 9 humans, 5 dogs, and 1 rodent, indicating circulation of the pathogen across multiple host species in the study area. Sequence similarity analysis of the representative amplicons showed 100% identity with Leptospira interrogans, and phylogenetic analysis further demonstrated that all study-derived sequences clustered within the pathogenic L. interrogans clade, with no clear host-specific segregation. The close clustering of sequences from humans, dogs, and rodent-associated samples suggests a shared transmission ecology or overlapping exposure network within this ecologically complex landscape. To our knowledge, this is among the first molecular evidence from Darjeeling demonstrating the occurrence of pathogenic L. interrogans across multiple host groups. These findings provide baseline molecular evidence of pathogenic L. interrogans in Darjeeling and support the need for broader One Health surveillance incorporating human, animal, rodent, and environmental sampling in the Eastern Himalayan region.