Background: Uropathogenic Escherichia coli is one of the most prevalent infectious agents in humans, but its resistance to commonly used antibiotics is growing rapidly. Objectives: The aim of this cross sectional study was to investigate the prevalence of tetracycline resistance determinants in urinary E. coli isolates obtained from patients in Iran. Methods: A total of 50 E. coli isolates from human urinary infections were characterized by cultural, biochemical, and molecular tests from 2014 to 2015. Isolates were tested for resistance to tetracycline by disc diffusion method. Then, the prevalence of tetracycline efflux genes (tetA, tetB, tetC, tetD, tetE, tetG, tetH and tetZ) was detected by means of molecular polymerase chain reaction method (PCR). Results: Of the 50 E. coli isolates tested, tetracycline resistance was identified in 62% of the strains. PCR analysis revealed that 36% of these isolates contained tetB gene, followed by tetA determinant with 32% frequency. The prevalence of tetG, tetZ, tetC, tetE, tetH, and tetD were 16%, 14%, 12%, 12%, 11%, and 8% among the isolates. Conclusions: Tetracycline resistance is widespread among uropathogenic E. coli isolates from human infections. Moreover, the distribution of tetracycline resistance determinants among the studied E. coli was very similar to the findings of the international sourced gallery of clinical E. coli Strains.