Purpose of this study was to determine the prevalence of antimicrobial resistance in microbial pathogens from different clinical samples. The current study was conducted in Lahore, Faisalabad,Rawalpindi and Islamabad as these cities are ranked as highly populated cities of Pakistan. Isolation and identification of pathogens from almost 750 diverse urine, blood and pus samples by aid of selected media cultures, gram staining and various biochemical tests. Isolated pathogens were checked for antimicrobial susceptibility patterns against frequently prescribed antibiotics. In present study, Gram-negative isolates were found in maximum percentages (80 Percent) while the remaining (20 Percent) were Gram-positives. The isolate which was most recurrently recognized from Gram-positives was S. aureus and most frequently identified isolate from Gram-negative was E. coli and P. aeruginosa. The prevalence of E. coli was 20 Percent in urine, 14Percentin blood, 12Percent in stool and 4 Percent in pus. Furthermore, the resistance rate of E. coli for Streptomycin, Nalidixicacid, Ciprofloxacin, Levofloxacin, Doxycycline, Moxifloxacin, Amikacin, Gentamycin, and Amoxicillin werebetween 12 Percent to a maximum of 100Percent. Gram positive S. aureus was found considerable in urine (9Percent), bloodand pus cultures. The resistance shown by the isolates ranged from 12Percent to 100Percent against ceftriaxone,ampicillin, chloramphenicol, doxycycline, co-trimoxazole, penicillin, erythromycin, vancomycin and tetracycline. Moreover, the antibiotics like Augmentin (71Percent) and Moxifloxacin (60Percent) showed highest resistance so these are considered as poor choices for treating S. aureus infections. We concluded from this study that strict legislation and better management techniques in context to use of antimicrobial for therapeutic purposes in human and animals can minimize the chances of development of antimicrobial resistance.