Australian canola (Brassica napus L.) has been relatively isolated from the global gene pool and limited knowledge is available for genetic variability based on DNA profiling. In the present study, genetic diversity of recent Australian canola cultivars was determined by simple sequence repeat (SSR) marker analysis. In total, 405 individuals from 48 varieties were genotyped with 18 primer pairs, resulting in 112 polymorphic features. The number of polymorphic features amplified by each SSR primer pair varied from 3 to 16. Analysis of molecular variance (AMOVA) detected 53.7% and 46.3% within- and between-cultivar variation, respectively. Intra-cultivar genetic variability differed according to cultivar. The number of polymorphic features per cultivar varied from 35 (Ag-Spectrum) to 72 (Ag-Insignia), while mean sum of squares (MSS) varied from 6.29 (Tornado TT) to 24.76 (Ag-Emblem). Genetic differentiation of cultivars generally reflected pedigree structure and origin by breeding organisation. Clustering and principal coordinate analysis (PCoA) indicated that the individuals were separated into 4 major groups. The genetic diversity information from this study will be useful for future Australian canola breeding programs.