Anchote (Coccinia abyssinica), is a perennial climbing, monoecious root crop, endemic and indigenous tornEthiopia. It is distributed over a wide range of agro-ecologies adapted to various altitudinal ranges. Thernroot of anchote stays for several years in the soil without being damaged and its shoot sprouts during rainyrnseason and gives fruit, then die out as the rain season ends. The root accumulates more food nutrients everyrngrowing season, and enters into dormancy until the next rainy season resumes.rnC. abyssinica is not only a valuable tuber food crop but also used as folklore medicines. Despite thesernimportances, there are very limited works available in literature regarding its genetics, agronomy,rnphylogeography, and evolutionary studies.rnIn this study, agro-morphological and molecular marker based genetic diversity and cytogeneticrncharacterization of anchote has been done. Data on 28 agro-morphological traits were collected under fiverndifferent experimental conditions, for a total of 182 accessions collected from southwestern part ofrnEthiopia. The results showed variations among and within accessions. On the basis of some traits, thernplants were grouped into few to several phenotypic classes. Deep green (Stcdg) and purple (Stcp) stemrncolors are observed as rare traits.rnIn molecular diversity study, a total of 47 EST-SSR markers were designed on watermelonâ€™s [Citrullusrnlanatus (Thunb.)] DNA sequences and only 13 of them amplified the target regions of which eight werernpolymorphic and the latter were used for diversity and population structuring analyses. Twenty four allelesrnwere observed across eight loci, where the number of alleles per locus ranged from 2 to 6, with an averagernof 3. Effective number of alleles ranged from 1.06 to 4.8 with an average of 1.93. Overall allelic frequencyrnper locus (0.007-0.967) revealed larger variations. Polymorphic information contents extracted per locusrnwere as low as 0.0619 and the largest was 0.76. Larger Shannon indices (average = 0.633) observed werernthe indication of better genetic diversity existing among anchote accessions. The top allelic richrnpopulations (Arjo-Leka Dulecha, Gimbi-Nejo, Abay Chomen-Bako Tibe and Dale Sadi-Dale wobera) arernleading accessions in genetic diversity parameter (He), but it is only Arjo-Leka Dulecha population thatrnpossesses 100% polymorphic loci. Although, no linkage disequilibrium was evident in this study, three locirn(WM-24, WM-34 and WM-29) showed significant deviation from Hardy-Weinberg equilibrium. The genetic diversity (He = 0.35, 0.06-0.79) estimated shows that there is a possibility of improvingrnanchote germplasm, especially by focusing on some accessions collected from Abay Chomen-Bako Tibe,rnDale Sadi-Dale Wobera, and Gimbi-Nejo, where both allelic richness and observed genetic diversity werernhigh. Anchote crop may be considered as panmictic population in which higher gene flow is common as itrnis observed in this study.rnAnalysis of molecular variance (AMOVA) revealed the highest proportion of genetic diversity withinrnindividuals (83.75%) and the least (7.87) among populations indicating high gene flows amongrnpopulations. Wider overall loci differentiation (FST = 0.01 to 0.3, with an average of 0.11) was observed.rnA range of 0.01-0.127 pair wise population differentiation was observed indicating that some populationsrnare very closely related, while others are somehow distant in kinship.rnCluster analyses based on EST-SSR data of different levels of anchote groups, i.e., populations, accessionsrnand individual samples did not show clear geographical patterns of origin, except for very few. In general,rnhowever, three apparent clusters were obtained. From neighbor joining tree (phylogram) Dale Wobera,rnGimbi, Metu, Yayo, Sayo, Gechi, Dhidhesa, and Shebe Sombo represent the older lineages (groups), whilernAbay Chomen and Leka Dulecha look recent population.rnAlthough, population structuring analysis of anchote accessions gave about maximum of seven sub-groupsrn(K = 7), there is no clear variation among the groups based on the allelic proportions of the sub-groups.rnSome populations were found to have experienced bottleneck. These include Sibu Sire-Wayu Tuka, GutornGida-Diga, Gera-Shebe Sombo, and Gumay-Goma.rnIn cytogenetic investigation, 2n = 20 chromosomes have been evidenced. All the chromosomes arernmetacentric in shape. No anchote cytotypes were recognized in this study.rnThe result of this study has many implications for breeding and conservation strategies, specially thernresults from cytogenetic characterization and EST-SSR based analyses. The morphological descriptors wernused may also contribute for further anchote description, variety development and improvement.rnKey Word: Anchote accession, Coccinia abyssinica, morphological trait, cytogenetics, EST-SSR, geneticrndiversity, population structure