Abstract: A total 72 drinking water sample were collected and analyzed by membrane filtration method during 1 year study from various points in Khairpur City . Out of these 58 (80.55%) samples were found to be contaminated with thermo-tolerant Escherichia coli 2. The susceptibility of these isolates to 35 antibiotics was studied by disc diffusion method and the organism was highly sensitive to levoflaxin, cefipime, enoxobid, noroxin, tarivid, ciproxin, avelox, amikacin, kanamycin, rocifin, pipenedic acid and slightly sensitive to cravit, naladixic acid, neomycin, cefizox, fortum cefotaxime, cefizox, fortum, tobramycin and cefoperoxone. The resistance against 16 antibiotics such as meropenem, linkomycin, fusidic acid, orbenin, penicillin, streptomycin, bacitracin, minocin, zinacef, amoxil, ceclor, claracid, cephalexin, augmentin, cephradin and dalacin was shown by these isolates. We report the presence of multi-drug resistance in thermo-tolerant Escherichia coli isolated in municipal water with different levels of prevalence in Khairpur City. In this study a higher number of positive results were obtained in all sampling points indicating the more fecally polluted municipal water.