Nigella sativa L. (commonly known as black cumin) belonging to family Rannunculaceae is an important medicinal plant with worldwide distribution. In Ethiopia, N. sativa occurs in all regions and agro-ecologies at different altitudinal ranges. This plant has a lot of importance in Ethiopia. However, there is no information available on molecular genetic diversity of this crop in respect to Ethiopia. Therefore, the aim of this study was to investigate the genetic diversity of black cumin populations collected from Amhara, Oromia, Tigray, Benshangul and South Nation's Nationality People regions of Ethiopia using ISSR marker. A total of 84 black cumin accessions were obtained from Institute of Biodiversity Conservation (IBC). The ISSR marker was used for computing gene diversity, percent polymorphism, Shannon diversity index and AMOVA. Overall, accessions from Oromia showed the highest gene diversity (H = 0.35) and Shannon information index (I= 0.52), followed by Amhara with gene diversity and Shannon index values of (0.35) and (0.51), respectively. NJ and UPGMA results showed strong grouping among accessions collected from the Oromia and Amhara region. The five geographical regions of Ethiopia showed different levels of genetic variation. Thus, conservation priority should be given for those regions that have low genetic diversity.