Strawberry plantlets of the ‘Earlibrite’ variety were irradiated with gamma rays to induce genetic variability for resistance to abiotic stress. This study aimed to identify the DNA polymorphism of ‘Earlibrite’ strawberry mutants using ISSR markers. For this purpose, a population of M1V0 strawberry plants was gamma-irradiated at 20 Gy. In addition, the irradiated plantlets were subcultured onto MS medium enriched with 0.13 M of 1-NAA and 2 M of 6-BAP. The genetic variation of the mutant was molecularly analyzed using the ISSR marker. Analysis was performed on M1V0 mutant plantlets. The results showed that 93.36% of 192 scoreable bands produced by the ISSR markers were polymorphic. An average of PIC values was 0.48 per primer with a range of 0.40 (ISSR_56) to 0.50 (ISSR_22, ISSR_55, ISSR_58, ISSR_60, ISSR_61, ISSR_62 and ISSR_L10). The primer MI ranged from 2.38 (ISSR_62) to 4.85 (ISSR_21). The genetic variation of mutant individuals ranged from 2.74% to 87.04% according to the Jaccard similarity index. Cluster analysis using ISSR markers grouped the ‘Earlibrite’ strawberry mutant genotypes into five distinct groups. It is concluded that 20 Gy gamma-ray induced mutation produces strawberry mutants and that ISSR was a valuable marker for mutant identification.