In this study, we analyzed the morphological, cytological, and molecular changes induced by different doses of ⁶⁰Co-γ radiation on root cuttings of the ground-grown chrysanthemum ‘Pinkling’ plants whose survival and height were decreased with increasing radiation dose. A variety of mutants were observed among irradiated ‘Pinkling’ plants; floral mutations included alteration of floral types, term ligulate floret number, capitulum diameter, and floral colour. Meiotic observation of mutants showed that the frequency of chromosome aberrations increased with radiation dose. The mutants were further analyzed using ISSR markers and on the basis of polymorphic DNA banding patterns, floral mutants were genotypically distinguished. Eighteen polymorphic ISSR primers produced 110 scorable bands, of which 93 (84.5%) were polymorphic. The percentage of polymorphism ranged from 60% to 100%. Jaccard’s dissimilarity coefficients varied from 0.337 to 1.000, which attested to the high genetic variability induced by irradiation. It is concluded that gamma ray irradiation induces a sufficient frequency of mutations and that ISSR analysis may offer a useful molecular marker for the detection of mutants in chrysanthemum. The present findings provide valuable information for future breeding of ‘Pinkling’.