A total of 325 clinical and environmental samples were collected from five hospitals in Baghdad from patients (Male and Female) of age range between (≤ 10-50) years in the period between 1/11/2012 to 7/1/2013. Out of 282 isolates, 56 identified as Klebsiella depending on cultural, microscopical and biochemical characteristics. The remaining isolates were identified as Escherichia coli, Staphylococcus aureus, Pseudomonas and Proteus, Staphylococcus epidermidis, Enterobacter and Shigella. Results of biochemical tests that was confirmed by using the API 20E and VITEK 2 system revealed that Klebsiella isolates (56 isolates) belong to four species: K. pneumoniae (40 isolates), K. oxytoca (3 isolates), K. terrigena (10 isolates) and K. ornitholytica (3 isolates). Thirty-six (90 %) of K. pneumoniae were isolated from clinical sources and 4 isolates (10 %) from environmental sources. K. pneumoniae isolated more frequently from urine sample and less from wound, ear swab, kitchen and bathrooms samples. Regarding to the patients gender, it was found that males had a tendency to get infection more than females when 19 (52.8 %) of patients were males and 17 (47.2 %) females. Moreover, the age group ≤ 10 were most subjected to the infection of K. pneumoniae. Antibiotic susceptibility of K. pneumoniae isolates against (15) of commonly used antibiotic was determined through disc-diffusion method. Results declared that, generally, the isolates were resistant to the antibiotics used except their sensitivity to imipenem. DNA was extracted from K. pneumoniae isolates. Results showed that the recorded range of DNA concentrations was 47.4-123.8 ng/μl and the DNA purity was (1.6-2.0). K. pneumoniae isolates was diagnosed molecularly by using polymerase chain reaction, 16S rRNA, rmpA and kfu genes were amplified and study the prevalence of the K serotypes, as well as, genes (except kfu gene) were amplified by using multiplex PCR. Results showed that all the isolates of K. pneumoniae gave a clear band with a molecular size 130 bp when PCR was performed with the primer that target the 16S rRNA. When using the primer specific for the capsule cluster gene magA and k2A, the result revealed that 23 (57.5 %) isolates belong to K1 serotype which gave a band of 1283 bp in size and 11 (27.5 %) isolates belong to K2 serotype which gave a band of 543 bp in size. These results suggest that magA and k2A genotype might be a useful marker to identify K1 and K2 serotypes of K. pneumoniae and these serotypes have been more prevalent than those that were neither K1 nor K2 (Non-K1/K2) (6 isolates (15 %)). The rmpA and kfu genes were amplified, the result stated that kfu gene is more frequent than rmpA gene, band was appeared of 797 bp in size represented Kfu gene in 30 of the isolates, while band was appeared of 536 bp in size represented rmpA gene in 11 of the isolates. Moreover, prevalence of kfu gene was in 21 isolates of serotype K1 which was more frequent than in serotype K2 (8 isolates) and Non-K1/K2 serotype (1 isolate), while rmpA gene was more frequent in the serotype K1 and K2 (5 isolates) than Non-K1/K2 serotype (1 isolates). Multiplex PCR was performed for K. pneumoniae isolates with four primers that target the 16S rRNA, magA, k2A and rmpA genes. Results showed that K. pneumoniae serotype K1 with rmpA positive isolates gave amplified bands for magA, rmpA and 16S rRNA genes, the K. pneumoniae serotype K2 with rmpA negative gave amplified bands for k2A and 16S rRNA genes.Moreover, K. pneumoniae serotype K1 with rmpA negative showed positive results with magA and 16S rRNA genes. Finally, K. pneumoniae Non-K1/K2 with rmpA negative showed only positive results with 16S rRNA gene. Multiplex PCR considered a reliable, relatively rapid, effective, easy application and repeatable and possible to be a powerful and potential tool for the routine clinical identification of Klebsiella species.