Abstract: Theheat shock protein 20(Hsp20) gene family plays an important role in regulating the stress response and plant development. The characteristics of Hsp20 in Cannabis sativa (CsHsp20), however, are still unclear. We systematically analyzed the CsHsp20 family based on the whole-genome and transcriptome database of Cannabis sativa using a series of bioinformatical tools. A total of 35 CsHsp20 genes (CsHsp20-1-CsHsp20-35) were identified in Cannabis sativa; they distribute onto 9 chromosomes and belong to 10 subfamilies, each with similar protein motifs. The promoter region of the CsHsp20 genes contains a variety of hormone-responsive and stress-responsive cis-elements, indicating that CsHsp20 genes are involved in plant growth and development and various stress responses. Protein interaction analysis showed that CsHsp20 proteins interacted with other members of the Hsp family and were regulated by transcription factors Hop and HSFA2. Transcriptome data showed that the expression levels of CsHsp20 genes were different among different tissues of Cannabis sativa and across different developmental stages. CsHsp20 genes were highly expressed mainly in hemp seed and its maturation stage, suggesting that CsHsp20 gene family members regulate the growth and development of hemp seed. Our research lays a foundation for the studying the function of CsHsp20 gene family and the directional cultivation of high-quality non-psychoactive medicinal cannabis.