Aims: The study was carried out firstly, to evaluate the prevalence of extended spectrum beta lactamase (ESBL), multidrug resistant Klebsiella pneumoniae isolates from Punjab, Pakistan and secondly, to characterize the genotypes of their beta lactamase producing enzymes and optimization of PCR based method for rapid and authentic detection of antibiotic resistant gene. Methodology and results: Two hundred of K. pneumonia strains were isolated from different clinical samples. Blood and MacConkey Agar were used to isolate and identify bacterial microorganisms while Muller Hinton Agar was used to evaluate the antimicrobial susceptibility against different antibiotics as per CLSI 2012 guidelines. ESBL producing bacteria were screened by double disk synergy /combination disk test. PCR was optimized and performed for resistant gene (CTX-M). The results showed that most of the isolates were resistant to multiple antibiotic including cephalosporin, aztreonam, sulphamethoxazole/trimethoprim, ciprofloxacin, doxycyclin and were sensitive to imipenam and amikacin. Frequency of CTX-M gene in ESBL producing K. pneumoniae was 94%. Conclusion, significance and impact of study: Based on the finding of this study it is suggested that prevalence of CTX-M gene (95%) is very high among ESBL producing isolates. Therefore, PCR based method may help clinicians for rapid detection and treatment of patients by choosing right medication against the resistant bacteria as early as possible.