AbstractFatty acyl-acyl thioesterases (FATs), which hydrolyze the thioester bond linking acyl chains to an acyl carrier protein, thereby terminating their elongation, contribute significantly to the fatty acid (FA) content and composition of seed storage lipids. The peanut (Arachis hypogaea L.) genome was found to harbor 21 FAT (AhFAT) genes, distributed over 12 of the 20 chromosomes. The length of their predicted translation products varied from 74 to 415 residues, and all but one included the 1–2 Acyl-ACP_TE conserved domains. All of the coding sequences were interrupted by at least one intron, with the exon number ranging from two to 12, and five of the genes were liable to alternative splicing. When the RNA-Seq platform was used to assess the transcriptional behavior of the 21 AhFAT genes, transcription of only 13 was detectable in samples of root, leaves, and developing seed; among these, six were transcribed throughout the plant, three were root-specific and one was leaf-specific. A detailed analysis of a pair of homologous AhFATs showed that the coding region of each was split into six exons and that both were transcribed in all of the plant organs surveyed (although the intensity of their transcription was not the same in immature seed). The product of both genes was deposited in the chloroplast outer membrane. The constitutive expression of these genes in either yeast or Arabidopsis thaliana increased the FA content, especially that of saturated FAs. In peanut genome, 21 AhFAT genes were found and two of them were transformed into yeast and Arabidopsis for function identification. Results showed that overexpression of these two genes could increase the FA content, especially the saturated FAs content.