Sesame (Sesamum indicum L.) is a nutritionally rich and economically important oilseed crop with considerable potential for genetic improvement. This review synthesizes current approaches for the characterization of sesame genotypes, integrating agro-morphological descriptors and chemical assays. Agro-morphological characterization remains a cornerstone for assessing genetic variability, encompassing key quantitative and qualitative traits such as plant height, branching pattern, days to flowering, capsule attributes, seed weight, and oil content. Substantial phenotypic variation observed within and among sesame populations highlights its utility in selection and breeding programs. However, reliance solely on morphological traits is constrained by environmental influences and the labor intensive nature of phenotypic evaluation.To enhance precision and efficiency in varietal identification, chemical assays including sodium hydroxide (NaOH), potassium hydroxide (KOH), gibberellic acid (GA₃) response, and 2,4-D soak tests—have been widely employed. These methods are rapid, reproducible, and effective for distinguishing genotypes, detecting admixtures, and classifying germplasm into distinct groups, thereby complementing morphological assessments. Furthermore, under the Protection of Plant Varieties and Farmers’ Rights (PPV&FR) Act, 2001, genotype registration is governed by Distinctiveness, Uniformity, and Stability (DUS) criteria, underscoring the importance of robust characterization frameworks. Comprehensive characterization and conservation of sesame germplasm are therefore imperative for safeguarding genetic resources and enabling their strategic utilization. Such efforts provide a critical foundation for parental selection and the development of high-yielding, stable, and climate resilient sesame varieties.