Mutation induction is considered as an effective way to enrich plant genetic variation, particularly for traits with a very low level of genetic variation. This research was conducted to assess genetic variation induced by gamma radiation in M2 and M3 mutant lines of canola (Brassica napus L.) by SSR and morphological characteristics and to identify useful mutants in terms of agronomic traits. Sixty-two mutant lines derived from gamma mutagenesis and their wild-type progenitors (‘RGS003’ and ‘Sarigol’ cvs) were used. Twenty-five polymorphic SSR primers were used in this study. Results of cluster analysis based on both morphological traits comprising plant height, days to flowering, days to maturity, number of pods/plant, number of seeds/pod, 1000-seed weight and seed yield/plant and SSR data revealed a separate grouping of mutant lines from control cultivars. SSR data analysis of mutant lines and controls demonstrated a considerable genetic variation among mutant lines, where 83% of primers generated polymorphic bands with 3.32 alleles per locus. The genetic distance calculated between mutant lines and their controls indicated a significant difference between mutant lines and controls. Although both morphological and SSR markers successfully discriminated mutant lines from controls, SSR primers could further discriminate between the mutant lines derived from the related cultivar. Mutant lines 24 derived from ‘RGS003’ and 16 and 26 from ‘Sarigol’ were considered as superior for breeding canola, which could be utilized in future genetic and breeding programs. Distinct classification of genotypes based on agromorphological and SSR data in the present study implies that morphological and SSR markers reflected different aspects of genetic variation among mutant lines.