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Background: Francisella tularensis could be disseminated through arthropod bites and exposure to infected animals, water, and aerosols. Water sources that are contaminated with rodent excrement could be a source of contamination; therefore, an analysis of water samples is an appropriate method to investigate the routes of dissemination. Since an outbreak occurred in one of the villages in East Azerbaijan. The current study aimed to investigate the Francisella isolation in the different water samples from East Azerbaijan, Iran. Sampling was carried out in East Azerbaijan province. Forty-six specimens of surface water were collected. Filtration, culture, and inoculation of the water sample into NMRI (Naval Medical Research Institute) inbreed mice were performed. DNA was extracted from filtered water samples, different organs of inoculated mice, and bacterial isolates and was tested by TaqMan real-time PCR by targeting ISFtu2 and fopA genes. Despite the unsuccessfulness in isolation of F. tularensis, molecular test results indicate the presence of bacteria in surface water. The highest rate of F. tularensis (ten from 46 water samples, 21.7%) was detected from injected mice based on molecular methods. Despite the high efforts of researchers to isolate Francisella spp. in Iran, in recent years, and also the evidence that shows the presence of this bacterium in different parts of the country, the culture was not successful again in this study and the molecular method still is recommended to identify the possible sources of Francisella spp., (Copyright © 2022. Published by Elsevier Ltd.)