The objective of this study was to determine and compare the fatty acid profile of milk fat, with particular reference to cis-9, trans-11 conjugated linoleic acid (CLA), in two indigenous Romanian sheep breeds (Spanca and Turcana), irrespective of the effects of diet and season. The ewes (n = 25 for each breed) received the same feed (total mixed rations) and were maintained under identical conditions. Four samples were collected from each individual ewe to determine fatty acids (FA), as methyl esters, using gas chromatography. The fatty acid groups, namely saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) acids were examined together with individual acids. Differences have been obtained between the two local breeds for the majority of FA. Fat in the milk of Turcana ewes has a healthier fatty acid profile than that of Spanca breed which has a lower concentration of SFA (60.9 vs. 73.3%) and higher concentrations of MUFA (31.0 vs. 21.0%) and PUFA (8.06 vs. 5.61%, respectively). Compared with the Spanca breed, fat in the milk of Turcana ewes had higher concentrations of trans-11 C18:1, C18:1 n9c, and cis-9 trans-11 CLA (2.67 vs. 1.01%, respectively). Δ9-desaturase activity is breed specific; vaccenic acid (trans-11 C18:1) desaturation and its conversion into cis-9, trans-11 CLA were more intense in the Turcana breed ewes. In conclusion, breed has a strong effect on the FA composition of ewe milk fat, and on cis-9, trans-11 CLA, in particular. The results obtained, support the sustainable use of animal genetic resources to improve the milk fat quality of ewes.