In this retrospective study of 332 cases of Legionnaires' disease (LD) in 4 Danish counties between 1995 and 2005 we aimed to compare the sensitivity of culture, PCR, urinary antigen testing, and serology to the mode of acquisition, serogroup, and severity of disease. Furthermore, we analyzed time to diagnosis. Laboratory confirmed cases of LD were found through the national Danish surveillance system and departments of clinical microbiology. In our study PCR was more sensitive (79.2%) than urinary antigen testing (70.4%), serology (54.8%), and culture (39.9%) (p<0.001).The sensitivity of Legionella urinary antigen test was higher among travel-associated cases (90.2%) compared to non-travel-associated cases (65.8%) and hospital acquired cases (45.7%) (p<0.001). Overall, the most common species and serogroup identified was L. pneumophila serogroup 1 (64.3% (110/171)). Community acquired cases with serogroup 1 were diagnosed earlier (mean 5 d, IQR: 4-9 d) than community acquired cases with other species or serogroups (mean 10.5 d, IQR: 5-19.5) (p<0.001). In conclusion, the urinary antigen test, PCR, and culture were conducive to the diagnosis of Legionella infection, and ordering of all 3 tests is recommended to ensure a definite and rapid diagnosis of Legionella.