Cannabis sativa L., a versatile plant grown for its seeds, fiber, and cannabinoids, has recently received significant scientific interest due to its wide range of industrial and pharmaceutical uses and economic prospects. The objective of this study is to assess the genetic variation of cannabis by examining its morphological and molecular characteristics in 83 different genotypes preserved at the Institute of Plant Breeding and Genetic Resources in Thessaloniki. Utilizing 10 microsatellite markers, important genetic variation was observed among the samples. Population structure analysis using STRUCTURE software indicated four distinct genetic subpopulations, which were further supported by the principal coordinate analysis (PCoA). The validity of these clusters was further confirmed through unweighted pair group method with arithmetic mean (UPGMA) phylogenetic analysis. The analysis of molecular variance (AMOVA) indicated that most of the genetic variation is present within individuals rather than among them or among populations (9% among populations, 53% within individuals). The expected heterozygosity (He or uHe) turned out to be 0.60-0.66, which supports moderate diversity. The fixation indices (Fst = 0.094, Fis = 0.417, Fit = 0.472) were statistically significant and suggested a moderate level of genetic differentiation between the two populations. Nei's genetic distance between hemp and marijuana groups was calculated as D = 0.288, indicating a moderate level of genome-wide divergence between these two major use types. Additionally, high-resolution melting (HRM) analysis utilizing sex-linked markers successfully identified 73 female and 10 male plants, underscoring the value of early sex identification in enhancing breeding strategies. These findings contribute valuable insights into the genetic diversity and sex determination mechanisms of Cannabis sativa and support future breeding programs which aim at developing cultivars with favorable traits.