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Summary Leptospirosis is a zoonotic disease that has spread worldwide. The primary origin of infection is rodents and wild animals that excrete Leptospira in their urine. This study was performed on 200 sheep in Meshginshahr to determine the serum prevalence of Leptospira infection. Serums were analyzed at a dilution of 1:50 and above on six Leptospira interrogans serovars, including Pomona, Conicola, Hardjo, Icterohemorrhagica, Australis, and Grippotyphosa and by using microscopic agglutination test (MAT). Among 200 samples, 22 samples were positive (11%), and 178 samples were negative (89%); There was a significant difference between positive and negative cases (P < 0.05). The most common strains were Icterohemorrhagica, and the lowest strains were Conicola and Pomona, and the Australis strain was not observed. There were five positive samples (13.51%) in the age group of 1.5 to 2 years old, ten positive samples (10.75%) in the age group of 2 to 4 years old, and seven positive samples (11.62%) in the age group of 4 to 6 years old. Also, a positive sample was not observed among the over six years old. One hundred fifty samples were related to ewe, and 50 samples were related to ram, that were positive 20 samples of ewe (13.33%) and two samples of ram (4%), there was a significant difference between different sexes in serum infection with Leptospira. The final result is that Leptospira infection is present in sheep in Meshginshahr city, and preventive measures should be taken to prevent its further spread.