Tetracycline resistance is increasing dramatically and represents a serious worldwide environmental issue. One hundred and eighteen Escherichia coli isolates from three different sources: chicken, human and treated sewage effluent (TSE) have been collected. Out of these 118 isolates, 65 E. coli strains (55%) were resistant to tetracycline. The resistant isolates have been identified by their protein patterns using Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI) Mass Spectrometry Biotyper and 16S rRNA sequencing both techniques confirmed the identification of E. coli isolates. Single and multiplex PCR have been applied to screen the isolates for 14 tet genes and found to be positive for 7 of them which are: tet (A, B, C, M, Q, W, 32). Five of the 7 positive genes tet (A, B, C, M, 32) have been sequenced successfully and their identity confirmed through BLAST @ NCBL Among the resistant isolates, tet A was the most detected gene (83%) followed by tet B (78.5%) and then tet 32 (38.5%). The rest tet determinants occurred at very low frequency. Many strains contained multiple determinants to resist tetracycline. Some strains contained 4 resistant genes tet (A/B/C/32) and tet (A/B/M/32) according to the literature the 4 tet gene combination is reported only in this study. The isolates showed a high variation of tet gene combination. The increased resistance of tetracycline and high variation in Tc genes is an indication of the abuse of this antibiotic and strict regulation needs to be implemented for control of its application.