The combination of trimethoprim-sulfamethoxazole is widely used for the prophylaxis and treatment of Pneumocystis pneumonia (PCP) caused by Pneumocystis jirovecii. Many studies have shown that mutations in the drug target, the dihydropteroate synthase (DHPS) gene, are presumably involved with the failure of prophylaxis and treatment. We have analyzed dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) mutations in P. jirovecii isolates recovered from Thai patients. Out of 17 samples, 11.7% (2) of the DHPS gene contained a double mutation at codon 55 and codon 57, whereas out of 18 samples, 61.1% (11) of the DHFR genes contained the silent mutation at codon 104. In comparison to previous reports, we have found a higher number of DHFR mutations but a lower prevalence of DHPS mutations.