Real-time quantitative polymerase chain reaction (qPCR) is a powerful tool for quantifying gene expression, combining both high sensitivity and specificity with efficient signal detection. Choosing the ‘correct’ normalisation control is essential and critical for obtaining reliable expression data. In this article, ten reference genes from Arachis hypogaea were analysed as internal controls for gene expression assay by using qPCR in two tissues of the seed, including embryo and cotyledon. The results showed that phospholipase D and actin were the most stable genes, whereas tubulin B4 and polyubiquitin 10 should be avoided when doing gene expression analysis because of their high variabilities in those tissue types. Although phospholipase D was scored as the most stable gene, it should be carefully validated for each particular experiment because it was easily regulated by different conditions. Taken together, actin was concluded as the optimal reference gene for studying gene expression in the mature peanut seed.