Klebsiella is an important human pathogen causing opportunistic nosocomial and community acquired infections. A major threat to public health concern regarding Klebsiella pneumoniae is the increasing incidence of multidrug-resistant strains. K. pneumoniae and K. oxytoca, are two clinically important Klebsiella species. The two clinically relevant species, are differentiated initially/preliminary by the ability to produce indole from tryptophan, K. oxytoca being indole positive. We report here the detailed biochemical and molecular analysis of Klebsiella isolates collected from sputum, urine, blood and environmental specimens. Where 75 clinical and environmental specimens' isolates were identified by biochemical tests. To differentiate K. pneumoniae from K. oxytoca, a multiplex polymerase chain reaction (PCR) was developed using species specific primers (rpo B gene for K. pneumoniae and of peh X gene for K. oxytoca). The position of bands on agarose gel, were at 108 bp for K. pneumoniae and 343 bp for K. oxytoca, confirmed strain identity. K. pneumoniae rpo B gene and K. oxytoca peh X gene of these isolates were determined by using Multiplex-PCR where, rpo B gene regions were determined in 74 (99%) of 75 Klebsiella strains while peh X gene region was determined in 1 (1%) of 75 Klebsiella strains. Results suggested that this newly developed PCR can be used as a diagnostic tool for correct identification and differentiation of K. pneumoniae and K. oxytoca, the two clinically important Klebsiella species. Antimicrobial test for these isolates indicated that all isolates are resistant to Penicillin and Clindamycin, 97.33% of isolates where resistance to Piperacillin and most of the isolates showed resistance to Cefoperazone and Ceftriaxone with a percentage of 94.67%. This study is important for determination the prevalence of Klebsiella infections in patients in Assiut University Hospital and in sewage water from different locations in Assiut Governorate and also, in this study the majority of isolates were multidrug- resistance to 16 antibiotics which confirms the serious (threat) of Klebsiella on human health.