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Hotel water systems colonized with <i>Legionella</i> spp. have been the source of travel-associated Legionnaires’ disease, and cases, clusters and outbreaks continue to be reported worldwide each year. A total of 132 hotels linked with travel-associated Legionnaires’ disease, as reported through the European Legionnaires’ Disease Surveillance Network, were inspected and tested for <i>Legionella</i> spp. during 2000–2019 by the public health authorities of the island of Crete (Greece). A total of 3311 samples were collected: 1885 (56.93%) from cold water supply systems, 1387 (41.89%) from hot water supply systems, 37 (1.12%) were swab samples and two (0.06%) were soil. Of those, 685 (20.69%), were collected from 83 (62.89%) hotels, testing positive (≥50 CFU/L) for <i>Legionella pneumophila)</i> serogroups 1–10, 12–14 and non-<i>pneumophila</i> species (<i>L. anisa, L. erythra, L. tusconensis, L. taurinensis, L. birminghamensis, L. rubrilucens, L. londiniesis, L. oakridgensis, L. santicrusis, L. brunensis, L. maceacherii</i>). The most frequently isolated <i>L. pneumophila</i> serogroups were 1 (27.92%) and 3 (17.08%). Significantly higher isolation rates were obtained from hot water supply systems (25.96%) versus cold water systems (16.98%) and swab samples (13.51%). A Relative Risk (R.R.) > 1 (<i>p</i> < 0.0001) was calculated for hot water temperature <55 °C (R.R.: 4.43), chlorine concentrations <0.2 mg/L (R.R.: 2.69), star ratings <4 (R.R.: 1.73) and absence of Water Safety Plan implementation (R.R.: 1.57).