Microsatellites are ubiquitous in genomes of various organisms. With the realization that they play roles in developmental and physiological processes, rather than exist as ‘junk’ DNA, microsatellites are receiving increasing attention. Next-generation sequencing allows acquisition of large-scale microsatellite information, and is especially useful for plants without reference genome sequences.

Paeonia ostii is an important woody oil plant cultivated in China on a large scale. Its seed oil is enriched with unsaturated fatty acids and a high content of alpha-linolenic acid (ALA), which are beneficial to human health. The aim of this research is to determine the qualitative traits characteristic of P. ostii seed from various production areas in China. In this study, seed quality traits were evaluated on the basis of proximate composition, content of fatty acids, tocopherol, secondary metabolites, and the antioxidant activity of seed coat (PSC) and kernel (PSK). A high content of total fatty acids (298.89–399.34 mg g⁻¹), crude protein (16.91%–22.73%), and total tocopherols (167.83–276.70 μg g⁻¹) were obtained from PSK. Significant differences were found in the content of palmitic acids (11.31–14.27 mg g⁻¹), stearic acids (2.42–4.24 mg g⁻¹), oleic acids (111.25–157.63 mg g⁻¹), linoleic acids (54.39–83.59 mg g⁻¹), and ALA (99.85–144.71 mg g⁻¹) in the 11 main production areas. Eight and seventeen compounds were detected in PSC and PSK, respectively. A significantly higher content of total phenols was observed in PSC (139.49 mg g⁻¹) compared with PSK (3.04 mg g⁻¹), which was positively related to antioxidant activity. This study indicates that seeds of P. ostii would be a good source of valuable oil and provides a basis for seed quality evaluation for the production of edible oil and potential ALA supplements from the promising woody oil plant.