There has been a growing prevalence of tetracycline-resistant bacteria across many species and genera. As a consequence, the efficacy of tetracycline treatment has diminished with time. So this work aimed to study of tetracycline-resistant genes among Enterobacter cloacae and Citrobacter spp. Out of a total of 364 clinical specimens, 135 (37.08%) exhibited bacterial growth. Out of the total, 22 (6.04%) were classified as gram-positive bacteria, 102 (28.02%) as gram-negative bacteria, and 11 (3.02%) as mixed growth (including both gram-negative and gram-positive bacteria). The Vitek-2 system findings indicated that the proportion of Enterobacter cloacae was 25 (6.86%), whereas Citrobacter spp. accounted for just 2 (0.54%) cases. The Enterobacter cloacae and Citrobacter isolates exhibited significant levels of resistance to several antibiotics, as shown by their drug susceptibility results. The PCR findings indicated that the tetrA gene was present in 8 out of 25 isolates (32%) of E. cloacae, and in 1 out of 2 isolates (50%) of Citrobacter spp. The tetrB gene was found in 92% of E. cloacae isolates (23 out of 25) and in 100% of Citrobacter spp. isolates (2 out of 2). In this study, the tetraC gene was identified in 6 out of 25 (24%) E. cloacae isolates and in 1 out of 2 (50%) Citrobacter spp. isolates. The TetraD and TetraE genes were not identified in the current study. The amino acid sequence analysis of the tetA and tetB genes revealed a similarity rate of 83% and 95% respectively.