The emergence of Pneumocystis jiroveci drug resistance has been suggested recently by the mutations in the gene encoding dihydropteroate synthase (DHPS). The aim of the present study was to determine the prevalence of DHPS mutations in P. jiroveci strains isolates from bronchoalveolar lavages (BAL) and sputum samples of 21 immunocompromised patients. We used the touchdown-PCR for amplification of DHPS gene and the restriction fragment length polymorphism (RFLP) technique for discrimination of wild and mutant DHPS genotypes. The DHPS amplification was positive in 17 patients (81%). The association of wild genotype and mutant genotype was detected in two patients after the enzymatic digestion of the PCR products by AccI and HaeIII. No mutations in the DHPS gene were seen in 15 patients. In addition, no variation was observed in DHPS genotypes detected in the repeated specimens (BAL and sputum) from some patients. The touchdown PCR-RFLP technique is a simple and rapid method for revelation of DHPS gene mutations in P. jiroveci strains. It could be advantageously used in clinical laboratory to control the prevalence of mutations associated with sulfa resistance.