Coffea arabica L., is indigenous to the highland of southwestern Ethiopia and southeasternrnSudan. It is the afromontane rain forest species of south west and south east part of Ethiopia andrngrown naturally at altitudes ranging from 1,300 and 1,800 meter above sea level. Humanrnpopulation pressure and land use change in and around the afromontane rain forest highlyrnthreatened the wild coffee populations of Ethiopia. Inter simple sequence repeats (ISSRs)rnmolecular markers were analysed. In the present study five wild populations of Coffea arabicarnfrom, Bench Maji-1(Maji), Bench Maji-2(Jeba), Dawero Banja, Limmu and Anfilo werernincluded. A total of 71 individual plants were analyzed using four ISSR primers, threerndinucleotides (810, 812,818) and one penta nucleotides (880) primers. A total of 30 clear andrnreproducible bands were amplified, and 100% were found to be polymorphic. The highestrnpolymorphic loci were revealed by primer 812 with 9 polymorphic loci. Among the populationsrnstudied, Bench Maji-1 and Dawero showed 100% polymorphism, while Limmu and Anfilornshowed moderate polymorphism of 56.67% and 63.33%, respectively, whereas Bench Maji-rn2showed the least polymorphism(30%). The average gene diversity relative to the overallrnpopulation was 0.45. AMOVA without grouping revealed that higher percentage of variationrn(79.4%) is attributed to the within population variation while the remaining variation is due tornthe among population variation (20.6%). Forest based UPGMA analysis based on Jaccardâ€™srncoefficient revealed one major group with Limu and Anfilo and two outliers (Bench Maji andrnDawero Banja). NJ analysis based on individuals of wild Coffea arabica showed two distinctrnmajor clusters and sub-clusters within both major clusters; and individuals from Bench Maji-1rnand Anfilo tend to form their own separate groups while others tends to speared all over the tree.rnIn 3D PCO, most of Dawero Banja and Bench Maji individuals were intermixed with each otherrnand made separate group from the other two populations but individual of Anfilo and Limmurnwere spread all over the plot.rnKey words: Coffee, Coffea arabica, genetic markers, genetic diversity and ISSR