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Hantavirus is a zoonosis transmitted from rodents to humans. Asymptomatic infected rodents can secrete hantaviruses in the urine, feces, and saliva. The main route of infection transmission to human is aerosols contaminated with the virus. This study was designed to evaluate the serological and molecular prevalence of hantavirus as an emerging zoonoses disease among street sweepers in Isfahan province, central Iran. Serum samples from 200 street sweepers in healthy condition and those with recent renal failure were tested by ELISA (IgM and IgG). Molecular analysis was subsequently applied for IgM positive cases. From these samples, 9 (4.5%) were positive, of which 2 (22.22%) were positive for both IgM and RT-PCR, while 7 (77.77%) were positive for IgG. The mean age and work experience of the positive cases were 39.7 and 11.5 respectively. According to our observations, all positive cases reported prevalence of rodents in their work place. The logistic regression test showed that the age and work experience were not risk factors for being positive, but prevalence of rodents in work place was a risk factor for being positive, when compared with negative cases. This is the first comprehensive study on the prevalence of hantavirus with positive results coming from Iranian population, which can raise the public awareness for the hantavirus infections as a public health threat.