Information on fatty acid (FA) profile is critical for the production and promotion of sheep milk and derivative dairy products. The presence of the essential ω-3 and ω-6 FA in milk fat as well as other less common FA, like linoleic acid isomers, has gained an increasing interest due to the consumer demand for a healthy diet. This research assesses the FA profile and estimates the lipid quality indices (ratio between hypocholesterolaemic and hypercholesterolaemic fatty acids, peroxidisability index, atherogenic index, and thrombogenic index) of raw milk and cream fat from two indigenous Greek sheep breeds (Karagouniko and Chios) at different lactation stages. Raw milk and cream fat presented a favorable ω-6/ω-3 ratio below 4:1. Atherogenic and thrombogenic indices of all studied milk fat fluctuated in sufficiently low levels (<3). The FA profile and lipid quality indices in both raw milk and cream samples differed significantly depending more on the lactation stage compared to the breed type. Raw milk fat from late lactation had more beneficial fatty acid profile compared to early and middle lactation stages. Differences among breeds were highlighted when raw milk and cream samples were compared within the same lactation stage. Raw milk and cream fat from Karagouniko breed were characterised by higher ω-3 proportion, lower ω-6/ω-3 ratio and lower thrombogenic index value compared to those from Chios breed.