Ten accessions of wild Borago officinalis were collected during missions in the course of a research activity carried out on plants producing nutraceutical compounds. The aim was to identify populations that produce a higher content of γ-linolenic acid, as this compound has important therapeutic properties and has been used in medical tests to treat rheumatoid arthritis, eczema, and psoriasis with good therapeutical results. The present study reports on the characterization of both the polyunsaturated fatty acid composition and on genetic diversity estimated by means of AFLP markers in several B. officinalis accessions collected in Southern Italy and grown in isolation by means of screen houses specifically designed for each single population. Polyunsaturated fatty acid were found in different amounts in all investigated accessions. The populations were very variable at the molecular level, and the analysis of relationships based on AFLP data, showed that similarity based on Jaccard’s index ranged between 0.330 and 0.742. Our investigations allowed us to identify a population that may be considered as a good source of GLA.