Background: Cannabis, an important industrial crop, has a high sensitivity to photoperiods. The flowering time of cannabis is one of its important agronomic traits, and has an important effect on its yield and quality. The CONSTANS-like (COL) gene plays a key role in the regulation of flowering in this plant. However, the specific biological and functional roles of the COL gene family in the cannabis is still unknown. Results: In this study, 13 CsCOL genes were identified in the cannabis genome. Phylogenetic analysis implied that the CsCOL proteins were divided into three subgroups, and each subgroups included conserved intron/exon structures and motifs. Chromosome distribution analysis showed that 13 CsCOL genes were unevenly distributed on 7 chromosomes, with chromosome 10 having the most CsCOL members. Colinearity analysis showed that two syntenic gene pairs of CsCOL4 and CsCOL11 were found in both rice and Gossypium raimondii. Among 13 CsCOLs, CsCOL6 and CsCOL12 were a pair of tandem duplicated genes, whereas CsCOL8 and CsCOL11 may have resulted from segmental duplication. Furthermore, tissue-specific expression showed that ten CsCOL genes were preferentially expressed in the leaves, one CsCOL in the stem, and two CsCOL in the female flower. Most CsCOL exhibited a diurnal oscillation pattern under different light treatment. Additionally, sequence analysis showed that CsCOL3 and CsCOL7 exhibited amino acid difference among the early-flowering cultivars and late flowering cultivars. Conclusion: This study provided insight into the potential functions studies of CsCOL genes, and highlight its roles in the regulation of flowering time in cannabis. Our results laid a foundation for the further elucidation of the functions of COLs in cannabis