In this study, we developed inter simple sequence repeat molecular markers to explore the genetic variation and relationships among 23 Polianthes tuberosa L. varieties. In all, seven primers were selected, and 99 identifiable amplicons were obtained, among which 89 were polymorphic with a polymorphic amplicon rate of 89.9%. The amplicons fragment ranged between 100 and 2200 bp. Jaccard’s coefficient was applied to calculate the similarity matrix between the varieties. The mean similarity coefficient among the 23 varieties was 0.64. Polianthes tuberosa ‘Double’ and P. tuberosa ‘Chia–Nong Pink Single’ had the farthest relationship, with a similarity coefficient of 0.35. By contrast, P. tuberosa ‘Chia–Nong Lady Jewel’ and P. hybrida ‘NCYU Pink Lady’ had the closest relationship, with a similarity coefficient of 0.8. A similarity coefficient of 0.606 was used as the basis for dividing the 23 varieties into three groups. Group (I), with a similarity coefficient of 0.678, comprised three subgroups and an independent variety. Group (II), with a similarity coefficient of 0.678, comprised three subgroups. Group (III) encompassed two varieties. P. tuberosa ‘Double’ is a variation of P. tuberosa ‘Single’ in Europe. According to the dendrogram, the two varieties were classified into the same group (II) and subgroup (II–a), implying that they were closely related. The results revealed that Polianthes tuberosa L. (hereafter referred to as “tuberose”) had rich genetic diversity, and the developed ISSR primers for tuberose had high discrimination ability. The study findings will facilitate subsequent tuberose genetic research and serve as a reference for further breeding and cultivation.
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