Streptococcus agalactiae is one of the uropathogens responsible for urinary tract infections (UTI) in children, pregnant women, and elderly people with chronic underlying diseases. This study was performed to determine the prevalence of urinary tract isolates of group B streptococci (GBS) in a group of females referred to a referral University Hospital in Iran. In this retrospective cross-sectional study, urine analysis and urine culture results of all female subjects referred to the laboratory of the Rasoul-e-Akram Hospital, Tehran, Iran in 2010 were reviewed. Bacteriuria, colony count, pyuria and demographic data of patients were also evaluated. In this study, 10,256 females were investigated; 2061 (20.1%) of them had positive urine cultures. GBS was the isolated microorganism in 184 (8.92%) cases, yielding a prevalence of 1.79% in total study population. The mean age of subjects with positive GBS cultures was 48.24 +/- 18.8 years, with a higher prevalence recorded in the 51-60 and 21-30 age groups. The highest rates of cultures positive for GBS were seen in December and January. GBS was found to be sensitive to the following antibiotics: cephalothin (100%), norfloxacin (96.9%), ampicillin (96%), nitrofurantoin (95.5%), and vancomycin (95%). In this study, GBS showed greatest resistance to tetracycline (81.6%) and co-trimoxazole (68.9%). In conclusion, the prevalence of GBS in females with suspected UTI is relatively low; however, attention to the age and susceptibility pattern of antibiotic treatment for UTI caused by this microorganism is necessary.