Borage (Borago officinalis L.) is a traditional vegetable grown and consumed in some Spanish regions. The objective of this study was to determine the variability and evolution of fatty acid composition in a borage germplasm collection formed by wild types, breeding lines, commercial varieties, and landraces. Fatty acids were analysed in petioles, the commonly edible part of the leaves, and the leaf blades, the by-product of the borage industry, in two growth stages: at the optimal harvest period (120 days after sowing) and at the end of the harvest period (150 days after sowing). The results showed that for each of the eight fatty acids identified, there were significant differences among the twelve borage genotypes depending on the developmental plant stage at sampling date and the part of the leaf analysed, the interaction effect also being statistically significant. The main polyunsaturated fatty acids identified were: linoleic acid (18:2 n6, LA), alpha-linolenic acid (18:3 n3, ALA), gamma-linolenic acid (18:3 n6, GLA), and stearidonic acid (SDA, 18:4, n-3), account for approximately 70% of polyunsaturated fatty acids. Blue-flowered genotypes differ from white-flowered genotypes by their high content of ALA and SDA, which can be exploited in borage breeding programs. Petioles from young plants present higher n6 fatty acids, while older plants produce a great amount of n3 fatty acids.