Background Previous literatures revealed that gamma rays have an increasing effect on salt tolerance in different plants. In vitro experiment was conducted to study the effect of gamma rays (20 Gray) on salt tolerance of four potato cultivars (Lady Rosetta, Diamante, Gold, and Santana). Results Gamma-treated Santana plantlets were more tolerant to salinity as compared to other cultivars. It showed a significant increment of fresh weight (250% over the untreated). Gamma-treated plantlets of Lady Rosetta, Diamante, and Gold showed higher activity of peroxidase (POD) and polyphenol oxidase (PPO). Isoenzymes analysis showed an absence of POD 3, 4, and 5 in Gold plantlets. The dye of most PODs and PPOs bands were denser (more active) in gamma-treated plantlets of Santana as compared to other cultivars. Both gamma-treated and untreated plantlets showed the absence of PPO1 in Lady Rosetta and Diamante, and PPO 3, 4, and 5 in Gold plantlets. Genetic marker analysis using ISSR with six different primers showed obvious unique negative and positive bands with different base pairs in mutant plantlets as compared to the control, according to primer sequence and potato genotype. The 14A primer was an efficient genetic marker between mutated and unmutated potato genotypes. Santana had a unique fingerprint in the 1430-pb site, which can be a selectable marker for the cultivar. An increment in genetic distance between Gold cultivar and others proved that the mutation was induced because of gamma rays. Conclusion We assume that irradiation of potato callus by 20-Gy gamma rays is an effective process for inducing salt resistance. However, this finding should be verified under field conditions. Graphic Abstract