Listeria monocytogenes is the causative agent of listeriosis, which may present as a severe disease with a high associated mortality rate of approximately 20%. Foodborne transmission accounts for almost all cases of illness. In this study 224 L. monocytogenes isolates from Australian clinical and non-clinical sources including food production, animal and environmental isolates were characterised. Serotyping, Multi-Locus Sequence Typing, and analysis of inlA gene sequence were performed. Serogroups IIA, IIB, and IVB comprised 94% of all isolates, with IVB overrepresented among clinical isolates. In contrast to this, IIA was the largest serogroup among dairy and meat sources. Lineage I isolates were most common among clinical isolates, and 52% of clinical isolates belonged to Sequence Type (ST) 1. Overall 39 STs were identified in this study, of which the largest were ST1 and ST3. These STs comprised 40% of the total isolates (n=90), and included isolates from clinical and non-clinical sources. ST204 was the third most common ST, and was found in a range of clinical and non-clinical sources. The high frequency of this subgroup has not been previously reported in similar studies in regions outside Australia. Two STs (ST121 and ST325) were found to contain premature stop codons in their inlA gene nucleotide sequence, an important virulence gene of the bacterium. This suggests these subgroups produce a truncated form of the InlA protein and may have reduced invasiveness. SNP subtyping showed differences in diversity when comparing isolates among individual STs. This study provides insights in the population structure of L. monocytogenes isolated from clinical and non-clinical sources in Australia, and can be used to inform food safety and public health efforts to control the bacterium.