Abstract: The MIKC-type MADS-box gene family plays an important role in flower development in plants. This study identifies members of the MIKC-type gene family in Cannabis sativa L. at the genome level, with the chromosomal location and linkage, evolutionary relationships, and identification of conserved motifs determined using bioinformatics tools. The results show that C. sativa contains 39 members of the MIKC-type MADS-box gene family (named CsMADS1-CsMADS39) unevenly distributed on nine chromosomes. The encoded proteins range in length from 146 to 503 amino acids, and the theoretical isoelectric points range from 5.19 to 10.12. Molecular weights range from 16 739.35 to 57 070.56 Da. The subcellular location of CsMADS genes is mainly in the nucleus. The result of conserved domain analysis showed that all genes contain the MADS conserved domain. The analysis of GO showed that all genes were annotated to 331 GO terms, which were clustered by molecular function. A phylogenetic tree showed that CsMADS genes could be divided into 14 subclasses, according to ABCDE homologous gene model; CsMADS genes are clustered with SQUA/AP1, AP3, PI, GGM13, AG, SEP/AGL2 subfamilies in Arabidopsis. There are abundant cis-acting elements in the upstream promoter region of CsMADS genes, mainly light regulatory elements. The results of real-time fluorescent quantitative PCR showed that some CsMADS genes are highly expressed in flowers and bracts, with tissue-specific expression. This study identified and analyzed the MIKC-type MADS-box gene family in C. sativa at the genome level, and provides a theoretical basis for further exploration of the function of MIKC-type genes and their role in regulating the growth and development of medicinally important hemp.