<p>Garden asparagus (<i>Asparagus officinali</i>s L.), a dioecious perennial crop, is highly valued for its nutritional and medicinal properties. The high similarity among cultivars and abundant intracultivar variation pose challenges to conventional cultivar identification methods. Leveraging the conserved parental genotype of ‘clone hybrid’ (CH) cultivars, this study employed a single-plant sampling strategy in which 20 individuals per cultivar were analysed to capture intracultivar variation. An innovative approach was further introduced for constructing a fingerprint database based on parental genotype data, which were reverse-inferred following Mendelian inheritance principles. Fifty EST-SSR markers, with an average polymorphism information content (PIC) of 0.6344, were selected to evaluate the genetic relationships among 62 cultivars. These cultivars were divided into three subgroups, revealing limited correlationt with their geographical origins and indicating their intricate genetic backgrounds. Furthermore, a core set of 7 EST-SSR markers effectively distinguished all cultivars except ‘Huamiao’ and ‘C₅-115’, which showed high similarity, suggesting potential synonymy. Validation with three identified cultivars and two novel hybrid combinations further confirmed the stability and accuracy of this fingerprint database. This study not only offers a reliable and practical tool for identifying the authenticity and purity of asparagus seeds but also provides a valuable foundation for efficient breeding programs.</p>