Leptospirosis is a zoonotic disease with a high incidence rate in many parts of the world due to the presence of various hosts for the pathogenic Leptospira. Tropical, subtropical and humid regions are suitable for long-term survival of the bacterium. Because of the temperate and humid climate, northern areas of Iran are suitable for pathogenic Leptospira and outbreak of the disease. Therefore, identification of infected areas is important from a public health and economic point of view. Previous studies show that the incidence rate of leptospirosis is increasing every year. Therefore, accurate diagnosis, control and prevention of this disease seem necessary through vaccination and raising public awareness, especially among high-risk groups. Today, diagnostic methods including immunofluorescence assay, enzyme-linked immunosorbent assay, microscopic agglutination test (MAT) and polymerase chain reaction (PCR) are used to diagnose the leptospirosis. MAT is the gold standard test for the diagnosis of leptospirosis with extensive applications in Iran. Due to the importance of this disease and its high prevalence in recent years, the present study aimed to investigate the epidemiology and diagnosis of leptospirosis in Iran.