<p>In Ethiopia, avocado trees have been grown for over 80&#xa0;years and have adapted to a wide range of topography, habitats, and climates; thus, the country is believed to harbor a high genetic diversity of avocados, but this has not yet been efficiently exploited. Hence, the primary aim of this study was to determine the extent and pattern of genetic diversity and population structure in avocado germplasm collections using single nucleotide polymorphism (SNP)&#xa0;markers. In our study, we genotyped a unique diversity panel of 267 avocado collections, which were collected from the first introduction center of avocados in Ethiopia (Sidama) and the Gedeo and Wolayta zones. The genomic DNA (50–100&#xa0;ng/ul) was extracted from young leaves using the NucleoMag^® Plant Extraction Kit. Next-generation sequencing was carried out using the Illumina HiSeq 2500 platform. A total of 2456 codominant SNPs were produced for the 267 avocado genotypes. Our results revealed that chromosome 1 had the highest number of mapped SNPs, whereas chromosomes 4, 10, and 11 had the lowest numbers of avocado references. The findings of this study showed that the average gene diversity, polymorphism information content, minor allelic frequency, and observed heterozygosity across all loci were 0.41, 0.32, 0.32, and 0.33, respectively, suggested the presence of a high genetic variation in avocado genotypes. Analysis of molecular variance (AMOVA) showed that only 6.55% of genetic variation existed among populations, 30.29% existed among individuals, and 63.16% existed within individuals. In addition, approximately 1.88%, 34.36%, and 63.76% of the molecular variance between altitudes, between samples within altitudes, and within&#xa0;samples, respectively, was based on the two altitudinal groups. Based on AMOVA, low- (F_ST = 0.02) and moderate-level genetic variations (F_ST = 0.07) were detected among the two altitudinal groups and studied populations, respectively, indicating the presence of a population structure. The Bayesian information criterion, discriminant analysis of principal components, and phylogenetic trees were grouped the studied avocado genotypes into five or six clusters. This high differentiation among germplasm suggests a significant genetic distance between these groups, which could pose challenges in breeding programs and offer opportunities for novel trait combinations. In conclusion, these findings fill gaps in our understanding of the molecular diversity and genetic structure of avocado germplasm and will serve as the foundation for future research on the development of crops to meet Ethiopian market demands.</p>