Objective: The aim of this study was to investigate the resistance mechanisms of carbapenem-resistant Enterobacteriaceae clinical strains recovered from Al Thawra University Hospital, Sana'a, Yemen. Methods: A total of 27 isolates showing decreased susceptibility to carbapenems were obtained from different clinical specimens in Al Thawra Hospital, Sana'a, Yemen. Strains were identified by Matrix Assisted Laser Desorption Ionization Time-Of-Flight spectroscopy. Susceptibility to antibiotics was determined by the disk diffusion method on Mueller Hinton agar. Carbapenemases-encoding genes, extended-spectrum β-lactamases (ESBLs), and plasmid-mediated quinolone resistance (PMQR) genes were screened by PCR. Bacterial isolates were typed by multilocus sequence typing (MLST). Results: Carbapenemase genes detection and sequencing showed that 18 (66.7%) isolates were Klebsiella pneumoniae (NDM-1, n = 13; NDM-1 + OXA-48, n = 3; OXA-48, n = 1; OXA-232, n = 1), 6 (22.2%) were Escherichia coli (NDM-5, n = 3; OXA-181, n = 2; OXA-48, n = 1), and 3 (11.1%) were Enterobacter cloacae (NDM-1, n = 1; OXA-181, n = 2). In addition the ESBL gene blaCTX-M-15 was detected in 14 K. pneumoniae and 2 E. coli isolates, and the blaCTX-M-216 was found in 1 E. coli isolate. Fifteen isolates were PMQR positive including qnrB1 (n = 1), qnrS1 (n = 5), qnrS4 (n = 2), and aac-(6')-Ib-cr (n = 7). The MLST typing showed a diversity of sequence type (ST) clones including Escherichia coli ST410 (3), ST448 (2), and ST648; Enterobacter cloacae ST78 and ST270; and Klebsiella pneumoniae ST395 (2), ST309, ST23, ST35, ST1728, ST15, ST231, and ST1428. Conclusion: This study reports the first description of OXA-48-like-producing Enterobacteriaceae and NDM-5 enzymes in E. coli in Yemen.