Pneumocystis jirovecii is an opportunistic fungus, which causes Pneumocystis pneumonia (PJP) in immunocompromised, COPD and TB positive patients with a high rate of colonization, morbidity and mortality. Dihydropteroate synthase (DHPS) gene mutations are well-reported in PJP. Although sulfa prophylaxis generally is associated with DHPS mutant infection, Multiple molecular techniques applied for detect sulfa resistance single-base mutation. Conformation sensitive gel electrophoresis (CSGE) is a rapid screening method for detection of DNA sequence variation, specifically single-base changes or small insertions and deletions. The current study is investigate on the DHPS sequence single-base dislocation among strains isolated from Iranian TB positive co-infected with PJP in association to increased levels of serum Lactate Dehydrogenase. Through high serum lactate dehydrogenase (LDH) levels have been associated with established Pneumocystis pneumonia. We investigated the DHPS mismatches in five P. jirovecii isolated of TB infected patients. For genetic identification of Pneumocystis isolates and detection of intraspecific variation, we developed a method for heteroduplex analysis. Our utilizing fragments was the DHPS gene regions, amplified by PCR method with specific primers. Serum LDH indicator was analysed for lung acute damages. In our results, at least 4 suspected isolates show more slowly migrating bands containing single heteroduplexes, reveal single-base mislocation in studied sequences. LDH level Peak was higher (p< 0.05) in patients with PCP (445± 155 U/L) in comparison patients with chronic TB (310±50 U/L). CSGE is a simple manual method, based on heteroduplex analysis, and compares well in terms of sensitivity with other screening technologies. Manual CSGE remains a low-cost, accessible, and effective approach for mutation screening, which can be carried out with minimal specialist equipment.