Point mutation of the dihydropteroate synthase gene causes Pneumocystis jirovecii resistance to sulfa drugs. The RFLP method was used to search for DHPS polymorphism in P. jirovecii strains isolated in Poland. Positive results of DHPS gene fragment amplification using nested PCR were achieved in 25 out of the 30 examined Pneumocystis DNA samples. Two (8%) of the 25 isolates had point mutation at codon 55 (Thr55Ala). The mutation-positive strains were obtained from HIV positive (1/15) and HIV negative (1/10) patients. The observed DHPS gene polymorphism may point to the appearance of P. jirovecii strains resistant to sulfa drugs in Poland.