Abstract Tree peony has been widely planted as an excellent oil crop with high proportion of α-linolenic acid (ALA) in its seeds. It was reported that pollen sources have clear effects on fatty acid (FA) composition of its kernels. Nevertheless, the molecular basis of this novel phenomenon is poorly understood. In this study, four Paeonia suffruticosa cultivars whose pollen caused significant differences in ALA content of Paeonia rockii were selected as pollen donors and grouped under high and low ALA groups. After pollination, total FAs content, composition and expression profiles of 10 genes involved in the FA and triacylglycerol (TAG) biosynthetic pathways were investigated during seed development. During the rapid oil accumulation in seeds, three desaturase genes (SAD, FAD2, FAD3) showed much higher expression in high ALA group, and their differential expression profiles corresponded well with the variations in ALA content among different pollen sources. Overall, pollen sources could affect the expression level of genes associated with fatty acid modification and subsequently affect the composition of FA in the kernels.