Malva sylvestris is an important medicinal plant in traditional and modern medicine. Various parts of this plant such as the leaves, flowers, roots, and seeds synthesize significant biological compounds used in traditional medicine and food industry. This research aimed to investigate the genetic diversity of M. sylvestris populations using SCoT markers and to determine significant marker-trait associations (MTAs). A field experiment was conducted using 10 populations from different regions of Iran using a randomized complete block design with three replications in 2023 in the research institute forest and rangeland, Tehran, Iran. Data collected for 18 morphological and biochemical traits. DNA was extracted from 50 genotypes belonging to using the CTAB method. Their genetic diversity was studied using 15 SCoT molecular markers. These markers amplified a total of 276 bands, of which 257 were polymorphic. On average, the number of bands produced by each primer for the 50 genotypes was 18.33, and the percentage of polymorphism was 86.68 %. The genetic distance between genotypes varied from 0.072 to 0.134 using the Nei coefficient. Genetic variation between and within the populations was estimated using analysis of molecular variance (AMOVA). Cluster analysis grouped the genotypes into five distinct groups. In addition, the study of population structure indicated the presence of five possible subgroups (K=5) within the studied populations, with fixation index values of 0.4106, 0.5121, 0.2166, 0.2607, and 0.0072, respectively. This study successfully identified significant marker-trait associations (MTAs) in M. sylvestris by using SCoT markers. The identified MTAs may breeding improve superior cultivars to enhance agronomic and biochemical traits.