Polymerase chain reaction analysis, direct DNA sequencing, and histological staining were used to determine whether Pneumocystis jirovecii was present in lung tissue specimens obtained, at autopsy, from 58 infants without identifiable immunodeficiency. The results of genotyping of these specimens were compared with the results of genotyping of specimens obtained from 384 human immunodeficiency virus (HIV)-infected adults with Pneumocystis pneumonia. P. jirovecii DNA was detected at the mitochondrial large subunit rRNA and dihydropteroate synthase loci in 100% and 53%, respectively, of the specimens obtained from infants. All specimens obtained from adults tested positive for P. jirovecii at both loci. Genotype distributions at both loci were significantly different in the 2 populations (P < .0001). The observation of different strains circulating in immunocompetent infants and HIV-infected adults suggests independent transmission cycles that warrant further study.