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Antimicrobial-resistant Klebsiella pneumoniae represent a global public health concern. K. pneumoniae strains isolated during 2010 and 2014-2016 within a single hospital of Molise Region, Central Italy, were analyzed testing antimicrobial susceptibility, clonality by pulsed-field gel electrophoresis (PFGE) and random amplified polymorphic DNA (RAPD)-PCR, and prevalence of carbapenem resistance genes by PCR. Forty isolates (23 wild-type in 2010 and 17 non-wild-type in 2014-2016) were collected from hospitalized patients (65.2 ± 18.1 years old, 75% male, 80% from intensive care unit-ICU). K. pneumoniae showed multidrug-resistant profiles and 15 resistotypes were identified (discriminatory power D = 0.88). The 69.6 and 17.4% of isolates in 2010 resulted intermediate and resistant to imipenem, respectively, and 91.3% was sensitive to meropenem, while 88.2% of isolates of 2014-2016 were resistant to both antibiotics. PFGE identified 16 clusters versus 23 by RAPD, 26 pulsotypes versus 33 RAPD patterns (D ≥ 0.97). PFGE separated strains according to isolation period and identified an outbreak occurred in the ICU during December 2014 and January 2015. No strains harbored bla GES , bla IMP , bla NDM-1 , and bla OXA-48 genes, as well as AmpC plasmid-mediated beta-lactamases genes. Only K. pneumoniae isolated during 2014-2016 were bla KPC positive. Prevalence of bla VIM was 87 and 76.5% during 2010 and 2014-2016, respectively. No strains colistin-resistant harbored mcr-1 plasmid-mediated resistance gene. The study findings underline an increased circulation of multidrug-resistant K. pneumoniae within the hospital, and the acquisition of carbapenem resistance mechanism. The implementation of surveillance and molecular characterization of isolates are needed to identify outbreaks, reduce the spread of resistance, and guide empirical therapy.