Isatis indigotica Fort. is a traditional medicinal plant, which has anti-inflammatory, antioxidant, and antiviral properties. Despite the development and licensing of several cultivars in recent years, morphological similarity among cultivars complicates their identification. The genetic diversity within I. indigotica significantly impacts the biosynthesis of bioactive substances. To elucidate genetic relationships and evaluate bioactive compounds, I. indigotica cultivars were analyzed using SSR markers. A total of 109 alleles were identified across 29 cultivars at 20 SSR loci, exhibiting a genetic diversity with an average polymorphic information content (PIC) of 0.46. Phylogenetic, principal coordinate analysis (PCoA), and Bayesian clustering revealed that genetic relationships were largely independent of geographic origin, potentially due to regional transplantations. Notably, some cultivars with distinct leaf sizes showed clear genetic differentiation, highlighting their potential as candidates for quality evaluation. A fingerprint was successfully constructed using five SSR markers. These findings provide technical support for cultivar identification, quality evaluation, and intellectual property protection of I. indigotica cultivars.