AbstractStructural variants (SVs) represent a major source of genetic diversity and are related to numerous agronomic traits and evolutionary events; however, their comprehensive identification and characterization in cucumber (Cucumis sativus L.) have been hindered by the lack of a high-quality pan-genome. Here, we report a graph-based cucumber pan-genome by analyzing twelve chromosome-scale genome assemblies. Genotyping of seven large chromosomal rearrangements based on the pan-genome provides useful information for use of wild accessions in breeding and genetic studies. A total of ~4.3 million genetic variants including 56,214 SVs are identified leveraging the chromosome-level assemblies. The pan-genome graph integrating both variant information and reference genome sequences aids the identification of SVs associated with agronomic traits, including warty fruits, flowering times and root growth, and enhances the understanding of cucumber trait evolution. The graph-based cucumber pan-genome and the identified genetic variants provide rich resources for future biological research and genomics-assisted breeding.