Pneumocystis jirovecii pneumonia (PcP) is an important opportunistic infection among immunocompromised patients. Genetic characterization of P. jirovecii isolated from HIV-positive patients, based on identification of multiple nucleotide sequences at eight distinct loci, was achieved by using PCR with DNA sequencing and RFLP. The present study showed that the mitochondrial large-subunit rRNA (mtLSU rRNA), the cytochrome b (CYB), the superoxide dismutase (SOD), the beta-tubulin (beta-tub), the dihydrofolate reductase (DHFR) and the dihydropteroate synthase (DHPS) loci sequences were more variable and therefore giving additional information than the thioredoxin reductase (Trr1) and the thymidylate synthase (TS) genes. Genotyping at those six most informative loci enabled the identification of 48 different P. jirovecii multilocus genotypes (MLGs). Significant statistical associations between infecting P. jirovecii genotypes and patients' age groups or PcP clinical status were found. Also, mtLSU rRNA sequences and specific genotypes from other three loci (CYB, SOD, and DHFR) were statistically associated. The results suggested large recombination between most P. jirovecii MLGs. However, one MLG occurred at a higher frequency than would be expected according to panmictic expectations, suggesting linkage disequilibrium and clonal propagation. The persistence of this specific MLG may be a consequence of clonal reproduction of this successful genotypic array in a P. jirovecii population with epidemic structure. The present study provided the description of multiple genomic regions of P. jirovecii, improving the understanding of genetic variability and frequency distribution of polymorphic genotypes, and exploring the criteria of clonality by testing over-representation of MLGs. Copyright © 2010 Elsevier B.V. All rights reserved.