Background Francisella tularensis is a bacterium causing the zoonosis tularemia. While many human infections are still caused by contact with infected animals, an increasing proportion occur through contact with a contaminated aquatic environment but the persistence of the bacteria in the environment remains unclear. Although this bacterium has been detected in the aquatic environment in several European country, the duration and conditions required for its persistence in water are not yet elucidated. The viable but non culturable (VBNC) state is a stress-resistant state where bacteria lose their ability to grow but can survive for prolonged time. Methods First, in vitro models have been constructed. An environmental water sample was sterilized by filtration, and inoculated with a clinical strain of F. tularensis subsp. holarctica, the subspecies causing tularemia in Europe. Six water survival models were then constructed in vitro, evaluating different incubation temperature (4, 18 and 37°C) and salinity levels (0 or 10g/L). Cultivability of F. tularensis was monitored by CFU counting and viability determination using propidium monoazid (PMA) qPCR and Live/Dead Bacligth™ assay for 4 months. Second, an environmental study by collecting surface water samples has been realized. The samples were taken from regions associated to the highest prevalence of tularemia cases in France in 2018. We investigated the presence of Francisella sp. and specifically F. tularensis subsp. holarctica in water by specific PCRs. Results Results of in vitro models showed that in low nutriment water incubated at 37°C the cultivability and viability of the bacteria dropped within a week, whereas the bacteria remained cultivable for 3 months at 4°C. Moreover, when cultivability finally declined upon incubation at 4 and 18°C, viability remained stable for more 10 weeks, suggesting a transition to the VBNC state. While cultivability decreased progressively, survival remained high and stable over the months, which suggests a progressive transition of the whole initial bacterial pool into the VBNC state. The environmental study was conducted in two sampling campaigns in West of France, in July 2019 and January 2020, allowing the collection of 87 water samples. Among 57 samples of the first campaign, 26% (15) were positive for Francisella sp. and 7% (4) for F. tularensis subsp. holarctica. The percentages were 83%, and 13% for the 30 samples of the second campaign. Among the 30 sites sampled during the two campaigns, nine were positive for Francisella sp. and one for F. tularensis subsp. holarctica both times. Conclusion Our work is the first demonstration of the ability of F. tularensis subsp. holarctica to persist in water in the VBNC state. While the bacteria progressively lose its capacity of cultivability in water over weeks, it however remains alive for a much longer period. Our environmental study reveals the aquatic presence and persistence of Francisella sp., including F. tularensis subsp. holarctica, in the studied regions of France. These results confirm that the aquatic environment can be a natural reservoir of F. tularensis surviving for months in water in a VBNC state and consequently being the potential source of tularemia outbreaks or sporadic cases. However, the factors allowing reactivation of the bacteria from the VBNC state into a virulent state remain yet to be identified.