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Genus Sophora L. belonging to family Fabaceae comprises about 80 species. It is a heterogeneous genus, ranging from tall trees to small, herbaceous plants. Morphological and genetic characteristics of two Sophora species, viz. S. secundiflora (Ortega) DC. and S. tomentosa L. were subjected to detailed study to delimit these species, where such data are lacking. During this study comparative morphology and anatomy of stem, leaves and petioles of both species was carried out. Moreover, DNA fingerprinting of both plants was carried out using 11 decamer random primers. The obtained results revealed that, many differences in morphological characters viz. the color of the flower, shape of the petiole and leaf dimensions. While the anatomical study reflecting other differential characters among them: the non-glandular hairs and the dimensions of some microelements. The features of the powdered stems and leaves are also described. Moreover, the DNA fingerprinting; created a total of 232 bands, out of the 99 were polymorphic representing a level of polymorphism of 42.67%. The primer (OPO-02) showed the highest degree of similarities (85.71%), while the lowest (35.29%) was recorded by the primer (OPO-10). This study concludes that the retrieved morphological, anatomical and genetic features provide a powerful identification and characterization tool for both of the studied Sophora species; even when used as powdered material. This study is a pioneer regarding the detailed botanical structure of both species; in addition to, the DNA fingerprinting to distinguish these species; in different prescriptions.