Simple SummaryThe biodiversity protection represents a challenge of the agenda ONU 2030 for Sustainable Development Goals (SDG). Autochthonous sheep breeds, including Altamurana, Bagnolese, Gentile di Puglia, Laticauda and Leccese, reared in continental Southern Italy, are strongly affected by extinction risk; thus, it is urgent to find new solutions to valorise their products and obtain sustainable and smart food from local farms. The objective of the present study was to assess the lamb carcass commercial quality, chemical and fatty acid composition of Altamurana, Bagnolese, Gentile di Puglia, Laticauda and Leccese autochtonous sheep breeds. Data on EU Mediterranean classification showed that the carcasses from both Bagnolese and Laticauda breeds were classified in the heaviest category C. Moreover, breed can influence the meat fatty acids profile, which is resulted rich in conjugated linoleic acid with good nutritional properties; thus, lambs’ meat from autochthonous sheep breeds can be considered interesting for human consumption. The present study can help to protect the biodiversity of livestock heritage of Southern Italy and open a new field for the valorisation and promotion of their derived products.In the present study, the evaluation of the carcasses and meat quality, in terms of chemical composition and fatty acid profile, of lambs from five autochthonous sheep breeds (Altamurana, Bagnolese, Gentile di Puglia, Laticauda, and Leccese) reared in continental Southern Italy, were studied. All the carcasses were evaluated according to the EU Mediterranean classification system for carcasses weighing less than 13 kg. Meat chemical composition and fatty acids profile were assessed on both loin and leg commercial cuts. Fatty acid composition of loin resulted in differences among breeds, displaying lower values of saturated fatty acid in Altamurana, Bagnolese, and Leccese breeds and the highest content of polyunsaturated fatty acid in the Altamurana breed. Principal component analysis grouped lamb according to fatty acid content and to conjugated linoleic acid (CLA), omega n-3 and n-6 fatty acids; thus, Altamurana, Bagnolese, and Leccese breeds are characterized by the highest values of CLA content. Our data demonstrated that lamb meat from autochthonous breed has good carcass quality and the content of CLA, n-3, and n-6 was valuable for human consumption; therefore, the valorisation of local meat quality can help to avoid the extinction of the autochthonous breed offering to the market and consumer’s high nutritive products.