AimsThis study aimed to investigate the prevalence of Simkania negevensis in the chlorinated water of spa swimming pools and domestic network systems. Methods and ResultsA total of 10 and 36 samples were collected from two domestic water supplies and four spa facilities, respectively. Simkania negevensis was isolated in Acanthamoeba castellanii monolayers and detected by Gimenez staining and immunofluorescence test. Simkania negevensisDNA, extracted from the positive samples, was confirmed by a nested PCR assay followed by sequencing. Simkania negevensis was detected in 413% of samples (domestic water: 50%; untreated spa supply water: 25%; chlorinated spa pool water: 429%) from all the examined water systems in successive samplings performed in 1year. The presence of S.negevensis was not correlated with the counts of Heterotrophic Bacteria and Pseudomonas aeruginosa. Unlike Legionella spp., which were never isolated from the swimming-pool water samples, S.negevensis was also detected in chlorinated spa water. ConclusionsThis investigation shows that Simkania is widespread in natural and man-made aquatic environments, which may represent possible sources of infection: in the swimming pools, in particular, the aerosol generated by the water movement could increase the risk of inhalation of infected particles. Significance and Impact of the StudyThis study represents the first evidence of the presence of S.negevensis in spa pool water. Sero-epidemiological surveys on spa users could help to clarify its transmission in this environment.