Cannabis sativa is a crop which has been cultivated since ancient times, with important cultural and industrial value. Due to its substantial economic impact, cannabis has attracted widespread scientific attention. A pan-genome is a significant tool for breeding, because it provides a comprehensive representation of genetic diversity. To provide a valuable tool for Cannabis breeding, we constructed a Cannabis pan-genome based on 113 accessions. A total of 24,679,380 bp of non-reference-genome sequences were assembled, identifying 1313 protein-coding genes. Using pan-genome analyses, a total of 32,428 gene presence/absence variations (PAVs) were obtained, and gene loss was recovered during the domestication of Cannabis. By partitioning the pan-genome using PAVs, a total of 23,309 core genes were identified, accounting for 71.88% of all genes in the pan-genome. In particular, there were 7148 flexible genes, making up 22.05% of the pan-genome. The flexible genes were associated with adaptive traits, including stress resistance and disease resistance in Cannabis. Population genetic analysis presented gene distribution, gene flow, and gene specificity on a pan-genome level. These results provide important genetic basis, functional genes, and guidance for Cannabis breeding.