Background:Pneumocystis jirovecii, formerly known as P. carinii, is an opportunistic fungus causing Pneumocystis carinii pneumonia especially in immunocompromised patients.Aim:The aim of this study was to detect P. jirovecii in sputum samples from patients suspected of having respiratory tract infections.Materials and Methods:In this study, 230 acid fast bacilli negative sputum samples from 230 patients presenting with respiratory tract infections submitted to three teaching hospitals’ medical microbiology laboratories in Osun and Oyo States, Nigeria for routine investigation were examined for P. jirovecii by microscopical and polymerase chain reaction methods.Results:P. jirovecii cysts were observed in 15 (6.5%) samples and polymerase chain reaction was positive for 29 (12.6%) samples out of 230 samples examined. It was observed that the detection of P. jirovecii was associated with age (P < 0.05) while there were no associations between diagnosis, sex, and prevalence of P. jirovecii (P > 0.05). Polymerase chain reaction was showed to be a better method for the detection of P. jirovecii based on the 51.7% sensitivity and 100% specificity of the microscopy.Conclusion:The study concluded that P. jirovecii is prevalent in patients with respiratory tract infections in hospitals from the southwestern part of Nigeria and should be included in diagnosis of these infections in this part of the world.