There are limited studies on characterization of Napier grass forage through the application of currently available advanced molecular techniques. Furthermore, ILRI genebank held collection of this forage grass from different part of the world with little information. Therefore, the objective of this study was to evaluate the extent of the genetic diversity of Napier grass accessions collected from different parts of the world and progeny plants raised from naturally produced seeds. The genomic DNA was extracted from leaves of 347 Napier grass genotypes and genotyped by the DArTseq platform. A total of 96,454 Silico DArT and 96,321 SNP markers were generated, of which highly informative 1001 SNP markers were selected for diversity analysis after screening. The average polymorphic information content (PIC) values of Silico DArT and SNP markers were 0.21 and 0.15 and Average heterozygosity of 0.26 and 0.18 respectively. Two major groups and ten sub-clusters were identified from STRUCTURE, Hierarchal clustering population stratification and diversity analysis. The sub-clusters obtained by cluster analysis were also confirmed through discriminant analysis of principal component (DAPC). Analysis of molecular variance (AMOVA was highly significant (P