The occurrence of tetracycline resistance determinants in Escherichia coli recovered from raw cow’s milk from the same farm sampled in two different time periods five years apart was investigated in this study. Detection of E. coli was performed and evaluated according to ISO 16649-2 and antibiotic resistance was screened by disk diffusion method. The polymerase chain reaction (PCR) was used to detect selected genes encoding resistance to tetracycline – tetA, tetB, tetC, tetD, and tetG. Of 89 samples of raw cow's milk, 84 (94.4%) were positive for E. coli. In total, 102 isolates were obtained. Fifty (49.0%) of these isolates were resistant to tetracycline. The most common gene detected in tetracycline-resistant isolates from 2010-2011was tetA (81.3 %), while tetB was most often (86.5%) found in isolates from 2005-2006. Neither the tetC, tetD, and tetG genes nor the co-occurrence of tetA and tetB genes were detected. None of the monitored genes was detected in two tetracycline-resistant isolates. Results of our study showed a significant shift in the distribution pattern of tetA and tetB genes in E. coli isolates from raw cow’s milk five years apart.