Abstract
Background: Oil-Camellia is an important woody oil crop, and most cultivated cultivars are hexaploid. Unresolved kinship among major cultivars and the lack of reliable identification methods have hindered both the development of new cultivars and the deployment of elite ones. An important reason is the limited availability of molecular markers suitable for genetic analysis. Results: Using five published oil-Camellia genomes, we surveyed genome-wide SSRs and found densities of one SSR per 4.22–4.67 kb across species. Based on the C. oleifera var. Nanyongensis reference genome, we designed 14,854 primer pairs and validated 132 new SSR markers. Genetic diversity and relationships among 44 hexaploid cultivars were assessed with a 30-marker subset, yielding 3–21 alleles per locus; polymorphism information content ranged from 0.07 to 0.91. Phylogenetic analyses using dosage-resolved SSR genotypes revealed the genetic relationships between 44 cultivars. We evaluated the discriminatory power of each marker and identified a minimal set (Coz09127 and Coz14197) that distinguishes all cultivars; using these two core markers, we constructed—for the first time—fingerprints for 44 hexaploid C. oleifera cultivars. Conclusions: We surveyed SSR distribution in oil-Camellia genomes and developed 132 SSR markers. Using 30 loci, we characterized genetic diversity and relationships among 44 hexaploid cultivars, and identified a minimal two-marker set that distinguishes all tested cultivars, enabling construction of cultivar DA-SSR fingerprints. These results strengthen the genetic toolkit for polyploid C. oleifera and support reliable cultivar identification and deployment in breeding and production. Supplementary Information: The online version contains supplementary material available at 10.1186/s12870-025-07601-8.