One of the major opportunistic pathogens in patients with burn injuries is Pseudomonas aeruginosa, which causes severe infections in burned patients. The objective of the study was to examine the molecular epidemiology of P. aeruginosa colonization in the burn unit of Shahid Motahari Hospital in Tehran, Iran. Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) analysis were employed to study 127 clinical and two environmental P. aeruginosa isolates collected from January to June 2008. In RFLP, the PCR products of 16S rRNA gene were digested with restriction enzyme Alu I, Hae III, and Rsa I, and the fragments generated were analyzed by agarose electrophoresis. Molecular typing by RFLP did show no discriminatory power for P. aeruginosa isolates, but RAPD-PCR revealed eight different genotypes; RAPD1to RAPD8 in clinical and environmental isolates. RAPD1 was the major genotype in clinical (n=64, 50.4%) and environmental isolates (n=1, 50%). The findings suggest that RAPD might have a superior typeability and discriminatory power over RFLP to study P. aeruginusa. Moreover, they highlight the need for further attention to the control of infection sources in Burn Units to prevent the transmission of the bacterium.