Klebsiella pneumoniae (K. pneumoniae) is a pathogenic bacteria responsible for a wide spectrum of infections in both hospital and community settings. A total of 66 isolates of K. pneumoniae were collected from different clinical sources in Palestine. The aim of this study was to determine the frequency of virulence genes in K. pneumoniae isolates using PCR technique, hypermucoviscosity (HMV) phenotype and antibiotic resistance profile. Rate of resistance to antibiotics was as follows: Trimethoprim/sulphamethoxazole (89%), Amoxicillin/clavulanic acid (82%), Aztreonam (77%), Tetracycline (71%), Ceftriaxone (67%), Imipenem (59%), Kanamycin (58%), Ceftazidime (56%), Levofloxacin (44%) and Ciprofloxacin (40%). In addition, isolates recovered from urine samples showed higher resistance (P < 0.05) against Imipenem, Ceftriaxone, Ceftazidime and Amoxicillin/clavulanic acid than isolates recovered from throat swabs. The prevalence of multidrug resistant K. pneumoniae isolates was 90.9%. Moreover, 5% of isolates were positive for HMV phenotype test. The prevalence of capsular polysaccharide genes among K. pneumoniae isolates was as follows: cps (100%), K1 serotype (21.1%), K2 serotype (11.7%), p-rmpA2 (15.2%), c-rmpA (7.6%), P-rmpA (12.1%), and magA (0.0%). The results of this study showed that 87% of rmpA genes are detected in non K1/K2 isolates and approximately 25% of tested isolates carried K1 serotype or K2 serotype or both serotype genes. Based on distribution of virulence factors, 3 (4.5%) strains were identified as probable hypervirulent K. pneumoniae. The presence of K1 or K2 or both serotype genes in these isolates together with other genes such as rmpA and high level of drug resistance should make bacteria a highly infectious agent, which leads to failure of treatment. Overall, this study demonstrates the significant role of rapid diagnosis and proper treatment of infections caused by this pathogen.