Hotel water systems colonized with Legionella spp. have been the source of travel-associated Legionnaires’ disease and cases, clusters or 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 Legionella spp. during 2000–2019 by the public health authorities of the island of Crete (Greece). A total of 3,311 samples were collected: 1,885 (56.93%) from cold water supply systems, 1,387 (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 (≥ 50CFU/L) for Legionella pneumophila) serogroups 1-10, 12-14 and non-pneumophila species (L. anisa, L. erythra, L. tusconensis, L. taurinensis, L. birminghamensis, L. rubrilucens, L. londiniesis, L. oakridgensis, L. santicrusis, L. brunensis, L. maceacherii). The most frequently isolated L. pneumophila 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 (p < 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 rating <4 (R.R.: 1.73) and absence of Water Safety Plan implementation (R.R.: 1.57).