Fifty-four carbapenem non-susceptible Klebsiella pneumoniae (CNSKP) isolates were collected from a Tunisian hospital over a period of 13 consecutive months. Carbapenemase production and the prevalence of carbapenemase-encoding genes were investigated using combined-disk test (CDT), modified Carba NP (mCarba NP) test, and UV-spectrophotometry method complemented by PCR experiments and sequencing. Carbapenemase production was detected by the mCarba NP test and CDT in 92.59% and 96.29% of the 54 CNSKP isolates, respectively; while imipenem hydrolysis was detected using UVspectrophotometry in the crude extracts of 44 isolates. bla NDM, bla OXA-48-like , and bla KPC carbapenemase-encoding genes were found in 48, 31, and 22 isolates, respectively. Remarkably, bla NDM-9, bla KPC-20 , and bla KPC-26 genes were reported. The co-occurrence of carbapenemase-encoding genes in a single isolate was detected in 62.96% of the isolates. The analysis of clonal relationships between the isolates by pulsed field gel electrophoresis revealed that the majority of them were genetically unrelated. Our investigation provides molecular data on enzymatic mechanism of carbapenem non-susceptibility among 54 CNSKP showing the dominance of bla NDM, and comprises the first identification of bla NDM-9, bla KPC-20 , and bla KPC-26 genes in a Tunisia hospital.