Pneumocystis jirovecii has been detected in lung tissue from patients with chronic obstructive pulmonary disease (COPD) and is associated with disease severity. The regional distribution of the organism in lungs is unknown, but differences in distribution of Pneumocystis could affect estimates of colonization prevalence. We examined the distribution of Pneumocystis in the lungs of 19 non-HIV-infected patients with COPD who were undergoing lung transplantation. DNA was extracted from explanted lungs. We found Pneumocystis colonization in lung tissue of 42.1% of patients with advanced COPD; however, there was significant regional variation in colonization between lung segments of individual patients. Colonization was detected more commonly in the lower and middle lobes than in the upper lobes. These findings suggest that single samples from an individual may underestimate the prevalence of Pneumocystis colonization and future studies may obtain a higher yield of Pneumocystis colonization detection when sampling the lower lobes.