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Background: C. Oleifera is among the world's largest four woody plants known for their edible oil production, yet the contribution rate of improved varieties is less than 20%. The species traditional breeding is lengthy cycle (20–30 years), occupation of land resources, high labor cost, and low accuracy and efficiency, which can be enhanced by molecular marker-assisted selection. However, the lack of high-quality molecular markers hinders the species genetic analysis and molecular breeding. Results: Through quantitative traits characterization, genetic diversity assessment, and association studies, we generated a selection population with wide genetic diversity, and identified five excellent high-yield parental combinations associated with four reliable high-yield ISSR markers. Early selection criteria were determined based on kernel fresh weight and cultivated 1-year seedling height, aided by the identification of these 4 ISSR markers. Specific assignment of selected individuals as paternal and maternal parents was made to capitalize on their unique attributes. Conclusions: Our results indicated that molecular markers-assisted breeding can effectively shorten, enhance selection accuracy and efficiency and facilitate the development of a new breeding system for C. oleifera. [ABSTRACT FROM AUTHOR]