Invasive listeriosis is a rare but serious foodborne disease that causes maternal-neonatal, central nervous system, and bloodstream infections. The aim of this study was to assess the whole-genome sequencing (WGS)-based genetic diversity of clinical Listeria monocytogenes isolates over a 7-year period and prove the effect of WGS application in food vehicle investigation. A total of 360 isolates were recovered during 2013 and 2019 through the national listeriosis special surveillance program. Two hundred twenty-six isolates (62.8%) were associated with pregnancy. All isolates belonged to lineage I (214 isolates) or lineage II (146 isolates), with 4 serogroups (46.9% IIb, 39.7% IIa, 12.5% IVb, and 0.8% IIc). All isolates were in 25 clonal complexes (CCs) and 3 singletons, with CC87, CC8, and CC5 being the most common causes of human listeriosis. All clinical isolates were positive for Listeria pathogenicity island 1 (LIPI-1), LIPI-3 was present in 21.4% of isolates and LIPI-4 was detected in 29.2% of isolates. LIPI-4-positive isolates, including CC87, sequence type (ST)619, ST382, CC4, and CC2, have been shown to confer hypervirulence. Fifteen isolates harbored at least one antimicrobial encoding gene, including tet (M), mef (A), msr (D), and dfr (G). The sublineage designations were consistent with CC designations, and 215 distinct cgMLST types (CTs) were classified, the most abundant being CT58 and CT750. In summary, there is a high level of genetic diversity among the clinical isolates. WGS has strengthened listeriosis surveillance and will be implemented for other foodborne bacteria in the National Molecular Tracing Network for Foodborne Disease.