Blackberry is a fruiting berry species with very high nutrient contents. With the recent increasing consumer demand for blackberries, new sources of germplasm and breeding techniques are required to improve blackberry production. This study was carried out to evaluate the genetic diversity (GD) and relationship among 55 blackberry (Rubus fruticosus) mutants derived from γ-ray treatment (52 lines) and N-methyl-N′-nitrosourea (MNU) treatment (three lines) using an inter-simple sequence repeat marker. A total of 18 bands were amplified with an average of 3.6 bands per primer. Among them, eight bands were identified to be polymorphic with a rate of 44.4%. In addition, the GD information content values were highest in the 60 Gy treatment population and the GD values were higher in the γ-ray treatment populations than in the MNU treatment population. According to a cluster analysis, all the mutant lines can be classified into five categories, and the genetic distance was greatest between the 80 Gy-irradiated population and other populations. These results indicate that mutant lines have high GD and can be effectively utilized for improving blackberry breeding.