Abstract: Background and Objective: Genetic diversity of two Sesbania species (annual and perennial) was studied via using different attributes karyotype formula, biochemical study (SDS-PAGE) and molecular studies (RAPD and ISSR markers). This study was focused on Sesbania due to its medicinal importance from the previous literature. The objective of the paper is studying the genetic conservation of Sesbania and put light on the main difference between annual and perennial one. Methodology: Seeds and young leaves of two species of Sesbania were collected from ten locations in Egypt and then the seeds prepared into slides via different steps of fixation and squashing. SDS-PAGE was done using the seeds of investigated taxa, in addition to two molecular markers RAPD and ISSR using five primers in each were used based on fresh young leaves of the investigated taxa. Results: This present investigation confirmed the chromosome number of two species of Sesbania (annual Sesbania sericea and perennial Sesbania sesban) was diploid 2n = 2x = 12. The karyotype parameters showed slight difference between ten taxa, where Sesbania sericea (Ssa5) had the lowest values of TF (%) and Syi index in addition to the highest value of A1, whereas, Sesbania sesban (Ssp1) had the highest values of TF (%) and Syi index and also the lowest value of A1. The SDS-PAGE analysis showed that two unique bands with molecular weight of 69 and 71 KDa were characteristic for annual Sesbania (Sesbania sericea), these two bands differentiate the annual species from the other studied perennial taxa. The RAPD and ISSR molecular markers yielded a total number of 57 bands out of which 31 were polymorphic ones and only 26 bands were found to be monomorphic. Conclusion: Karyological parameters beside biochemical and molecular attributes had the ability to study the genetic diversity of Sesbania species and can be used in the conservation of these species in gene banks.