Fungi of the genus <i>Pneumocystis</i> are obligate parasites that colonize mammals' lungs and are host species specific. <i>Pneumocystis jirovecii</i> and <i>Pneumocystis carinii</i> infect, respectively, humans and rats. They can turn into opportunistic pathogens in immunosuppressed hosts, causing severe pneumonia. Their cell cycle is poorly known, mainly because of the absence of an established method of culture <i>in vitro</i> It is thought to include both asexual and sexual phases. Comparative genomic analysis suggested that their mode of sexual reproduction is primary homothallism involving a single mating type ( <i>MAT</i> ) locus encompassing plus and minus genes ( <i>matMc</i> , <i>matMi</i> , and <i>matPi</i> ; Almeida et al., mBio 6:e02250-14, 2015). Thus, each strain would be capable of sexual reproduction alone (self-fertility). However, this is a working hypothesis derived from computational analyses that is, in addition, based on the genome sequences of single isolates. Here, we tested this hypothesis in the wet laboratory. The function of the <i>P. jirovecii</i> and <i>P. carinii matMc</i> genes was ascertained by restoration of sporulation in the corresponding mutant of fission yeast. Using PCR, we found the same single <i>MAT</i> locus in all <i>P. jirovecii</i> isolates and showed that all three <i>MAT</i> genes are often concomitantly expressed during pneumonia. Extensive homology searches did not identify other types of <i>MAT</i> transcription factors in the genomes or <i>cis</i> -acting motifs flanking the <i>MAT</i> locus that could have been involved in <i>MAT</i> switching or silencing. Our observations suggest that <i>Pneumocystis</i> sexuality through primary homothallism is obligate within host lungs to complete the cell cycle, i.e., produce asci necessary for airborne transmission to new hosts. <b>IMPORTANCE</b> Fungi of the genus <i>Pneumocystis</i> colonize the lungs of mammals. In immunosuppressed human hosts, <i>Pneumocystis jirovecii</i> may cause severe pneumonia that can be fatal. This disease is one of the most frequent life-threatening invasive fungal infections in humans. The analysis of the genome sequences of these uncultivable pathogens suggested that their sexual reproduction involves a single partner (self-fertilization). Here, we report laboratory experiments that support this hypothesis. The function of the three genes responsible for sexual differentiation was ascertained by the restoration of sexual reproduction in the corresponding mutant of another fungus. As predicted by self-fertilization, all <i>P. jirovecii</i> isolates harbored the same three genes that were often concomitantly expressed within human lungs during infection. Our observations suggest that the sexuality of these pathogens relies on the self-fertility of each isolate and is obligate within host lungs to complete the cell cycle and allow dissemination of the fungus to new hosts.