Alpha-linolenic acid (ALA, C18:3) is an essential fatty acid integral to human growth and development. Despite its significance, the ALA content in peanut seeds-a major global oilseed crop-is notably low. This study employed bioinformatics analysis, tissue expression, and promoter function evaluations to investigate AhFAD3, which encodes the microsomal omega-3 fatty acid desaturase that is directly responsible for ALA accumulation through converting linoleic acid (LA) to ALA. We identified the active AhFAD3 gene, AhFAD3-A01, with the functional protein encoded by AhFAD3-A01 localized in the endoplasmic reticulum (ER) and found to be pivotal in ALA synthesis in seeds. The low expression of AhFAD3 genes during the late stages of seed development, coupled with the specific activation by only AhFAD3-A01 and AhFAD3-B01 promoters in seeds, results in the low ALA levels in mature peanut seeds. To enhance ALA content, the constitutive promoter CaMV35S and the seed-specific promoter P