Bacterial infections represent a very serious problem that threatens human health, antibiotics were designed to attack the causative agents of infectious diseases, but some bacterial pathogens became virulent and resistant to antibiotics by different mechanisms, resistance genes represented one of those mechanisms. This study attempts to screen the existence of five different resistance genes (mecA, TEM, FemA, MexD, and AmpC) among 25 bacterial isolates divided into two groups the first was non-clinical bacterial type strains including Bacillus subtilis, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella typhi, and the other group includes some clinical bacterial isolates. Evaluation of their susceptibilities to different 12 antibiotic discs and attempting to find the relationship between genotype and phenotype assessment. Different responses were reported which varied from slightly susceptible to multidrug-resistant such as P. aeruginosa and K. pneumonia which could be considered as multidrug-resistant strains. Therefore, detection of resistance gene became crucial and critical to recognize the mechanism of resistance, five pairs of primers were included to investigate five responsible genes belonging to beta-lactamases, efflux pump, and methicillin resistance. Conclusively, the PCR technique is a very accurate tool to check the genetic resistance whether being expressed to phenotype or not. Moreover, the clinical bacterial isolates appeared more resistant that reflecting the impact of the surrounding environment on bacterial behavior.