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In Iran, leptospirosis is endemic to Caspian Sea littoral. The disease appears as a seasonal infection mostly affecting people in rural areas involved in farming. We investigated the prevalence of this infection among suspected patients in Gilan Province by an indirect immunofluorescent assay (IFA), and two PCR protocols, a nested-PCR and a real-time PCR (qPCR), targeting rrs and lipL32 genes, respectively. We also identified the common Leptospira species by sequencing a partial sequence of rrs gene. Out of the 128 sera examined by IFA, 25.78% were positive with the antibody titers ≥1/80. The antibody titer in 39.06% of sera ranged from 1/10 to 1/140, and 35. 16% showed no antibodies, all considered negative. Nested PCR and qPCR detected Leptospira DNA in 20.31% and 18.75% of the sera, respectively. The two PCR assays had 98.43% agreement (K = 0.93) and showed an inverse correlation with the IFA titers. Also, three pathogenic Leptospira species, L. kirschneri (n = 10), L. introgans (n = 8), and L. borgpetersenii (n = 2) were identified from the clinical specimens in the study area. In our hands both PCR assays proved very efficient for early diagnosis of illness and could be used in combination with IFA for both diagnosis and epidemiological studies, but nested PCR was cheaper and appeared more appropriate for our laboratories in rural settings., (Copyright © 2018 Elsevier B.V. All rights reserved.)