For centuries, crop improvement has served as the basis of food security of ever increasing human population. Though vast germplasm collections are available; their exploitation for crop improvement still depends upon efficient assessment of genetic diversity. Genetic variability is the key element in adaptation of plants to varying climates. While crops with narrow genetic diversity are vulnerable to stresses. The estimation of extent and pattern of genetic variability is a prerequisite for generating superior varieties. Genetic diversity analysis generates key information to dissect genetic variations in crop germplasm with the help of morphometrical, biochemical and molecular tools. Among these, DNA markers provide a reliable and detailed insight into the similarities and differences among crops. In this chapter, we discuss the applications of phenotypic and molecular markers to probe genetic divergence in crops and present case studies that describe the significance of these tools to characterize sorghum germplasm. Furthermore, we spotlight sorghum biodiversity exploration efforts worldwide and propose future directions.