Abstract: Objective To analyze the molecular characteristics of 23 clinically isolated Listeria monocytogenes strains. Methods A total of 23 Listeria monocytogenes strains isolated from Nanjing Drum Tower Hospital from March 2013 to May 2021 were collected. The virulence genes，drug resistance genes, genealogy, sequence analysis (ST) and clonal complexes ( CCs) of the strains were analyzed by whole genome sequencing technology. Serotyping was carried out by mutiplex PCR. Antimicrobial susceptible test were carried out by E-Test and disk difusion test. Results The 23 strains belonged to 2 lineages, mainly lineage I with 12 (52.2% ) strains and was divided into 8 ST types, of which ST8 was the dominant type accounting for 39. 1% . The genotypes included 7 CC types ( CC87, CC1, CC3, CC5, CC224, CC11 and CC8) among which the dominant CC type was CC8 with 9 strains ( 39.1%). All the isolates carried Listeria virulence island LIPI-I (ActA, hlyA, mpl, plcA, plcB and prfA) and inlA, inlB genes. The fosX resistance genes were detected in all the 23 strains , of which one strain carried both tet( M) and dfG resistance genes. The serogroups of the 23 strains were divided into 1/2a, 1/2b and 4b, mainly 1/2a, with 13 strains (56.5% ). All the strains were susceptible to 9 antimicrobials including penicillin, ampicillin, meropenem and vancomycin, and only 1 strain was resistant to cotrimoxazole and tetracycline. Conclusion The clinically isolated Listeria monocytogenes showed low drug resistance and genetic diversity. A variety of virulence genes carried by these strains should be the important factors in their pathogenicity.