Mycoplasma hominis is normally found in the urinary tract of human and its role in Urinary Tract Infection (UTI) has been proved. This bacterium causes inflammatory responses and accumulation of leucocytes in urethra. In spite of the presence of the bacteria, the urine culture might be negative. Mycoplasma hominis can be transferred sexually and causes human infertility. The present study was conducted to detect and identify Mycoplasma hominis by molecular methods in urine samples of the patients with UTI, who were referred to our hospital. A total number of 864 urine samples from the patients with UTI were subjected to this study. After routine culture, urine analysis were performed on the samples. The DNA was extracted from the sediments of the urine samples, using phenol and chloroform method. Polymerase Chain Reaction (PCR) was conducted on the extracted DNA to detect the 16S rRNA of Mycoplasma hominis, with the primers; RNAH1 and RNAH2. Based on the results of PCR tests, out of 100 pyuria positive samples, 9 and 1% were infected with Mycoplasma sp. and Mycoplasma hominis, respectively. The sequencing of amplified product of 16S-rRNA revealed a single nucleotide substitution (269 T A), compared with the reference gene of this species.