Background: Escherichia coli (E. coli) is a major cause of urinary tract infection in children and women. Several virulence factors have been detected in uropathogenic Escherichia coli strains. P, S and Afa fimbria are the most important factors which are coding by pap, sfa and afa genes, respectively. This study aimed to determine the presence and prevalence of fimbrial virulence genes in Escherichia coli isolates from patients with urinary tract infection in Rafsanjan city, Iran. Methods: In this study, 145 urine samples were cultured for identification of Escherichia coli. The isolates were examined via standard biochemical tests. DNA extracts from the confirmed isolates were examined to determine papEF, afaBC and sfa / focDE genes using multiplex-polymerase chain reaction (PCR) method. Findings: Among the cultured samples, 122 Escherichia coli isolates were determined. Virulence genes genotyping of isolates showed that 38.50% of the isolates were possessed at least one of the three virulence genes. The papEF gene was the most prevalent (18.85%) and prevalences of sfa/focDE and afaBC were 2.45% and 0.81%, respectively. Twenty isolates (16.39%) contained both papEF and sfa/focDE genes. Conclusion: The results of this study in comparison with other researchers showed that papEF gene is in the high frequency, and then, afaBC and sfa/focDE fimbrial genes.