Structural variations (SVs) are major genetic variants that can be involved in the origin, adaptation and domestication of species. However, the identification and characterization of SVs in Spinacia species are rare due to the lack of a pan-genome. Here, we report eight chromosome-scale assemblies of cultivated spinach and its two wild species. After integration with five existing assemblies, we constructed a comprehensive Spinacia pan-genome and identified 193 661 pan-SVs, which were genotyped in 452 Spinacia accessions. Our pan-SVs enabled genome-wide association study identified signals associated with sex and clarified the evolutionary direction of spinach. Most sex-linked SVs (86%) were biased to occur on the Y chromosome during the evolution of the sex-linked region, resulting in reduced Y-linked gene expression. The frequency of pan-SVs among Spinacia accessions further illustrated the contribution of these SVs to domestication, such as bolting time and seed dormancy. Furthermore, compared with SNPs, pan-SVs act as efficient variants in genomic selection (GS) because of their ability to capture missing heritability information and higher prediction accuracy. Overall, this study provides a valuable resource for spinach genomics and highlights the potential utility of pan-SV in crop improvement and breeding programmes.