To determine routes of colonization and genotypic variation of Pseudomonas aeruginosa leading to ventilator-associated pneumonia, colonization of the rectum, stomach, oropharynx, and trachea was studied chronologically in 10 patients. Ninety-one isolates of P aeruginosa were genotyped; seven different genotypes were identified. Patients developing ventilator-associated pneumonia caused by P aeruginosa were colonized at multiple body sites and may be colonized with multiple genotypes. The upper respiratory tract is the predominant initial site of colonization with P aeruginosa.