Sixteen broad-spectrum cephalosporin-resistant Klebsiella pneumoniae isolates were recovered between April and June 2013 from Palestinian hospitals, Gaza Strip. Genes encoding extended-spectrum beta-lactamases (ESBLs) and other resistance genes were characterized by polymerase chain reaction and sequencing. The following β-lactamase genes were detected: bla+ bla+ bla (six isolates), bla+ bla+ bla (two isolates), bla (two isolates), bla+ bla (one isolate), bla+ bla (one isolate), bla+ bla+ bla(one isolate), bla+ bla (one isolate), bla+ bla (one isolate), and bla (one isolate). The ISEcp1 (in four cases truncated by IS26), orf477, or IS903 sequences were found upstream or downstream of bla genes. The aac(6')-Ib-cr gene was found in seven isolates. The qnrS1 and qnrB1 genes were detected in five isolates and two isolates, respectively. Seven isolates contained class 1 integrons with four gene cassette arrangements: dfrA5 (three isolates), dfrA12-orf-aadA2 (two isolates), dfrA17-aadA5 (one isolate), and aadA1 (one isolate). A high clonal diversity was also observed among studied isolates by pulsed-field gel electrophoresis (12 unrelated profiles). This study demonstrates for the first time the emergence and polyclonal spread of multidrug-resistant ESBL-producing K. pneumoniae isolates among patients in a hospital setting in Gaza Strip, Palestine.