SummaryTo enrich our knowledge about gene network of fatty acid biosynthesis in cottonseed, we conducted comparative transcriptome to reveal the differences in gene expression between Gossypium hirsutum and Gossypium barbadense during cottonseed development. The prolonged expression period and increased expression abundance of oil‐related genes are the main reasons for producing high seed oil content (SOC) in G. barbadense, which manifested as the bias of homeologous gene expression in Dt‐subgenome after 25 day postanthesis (DPA). The dynamic expression profile showed that SAD6 and FATA are more important for oil biosynthesis in G. barbadense than that in G. hirsutum. Three key transcription factors, WRI1, NF‐YB6 and DPBF2, showed their elite roles in regulating seed oil in cotton. We observed that sequence variations in the promoter region of BCCP2 genes might contribute to its divergence in expression level between the two species. Based on the quantitative trait loci (QTL) information of the seed oil content and utilizing additional G. barbadense introgression lines (ILs), we propose 21 candidate genes on the basis of their differential expression level, of which the GbSWEET and the GbACBP6 showed the potential functional to improve the oil content. Taken together, studying the different expression of oil‐related genes and their genetic regulation mechanisms between G. hirsutum and G. barbadense provide new insights to understanding the mechanism of fatty acid biosynthesis network and fatty acid genetic improving breeding in cotton.