Energy supplementation can alter ruminal biohydrogenation (BH) of polyunsaturated fatty acids, producing diverse intermediates which may lead to different milk fatty acid (FA) composition. The aim of the study was to evaluate rumen FA profile in dairy ewes supplemented with contrasting sources of carbohydrates on a fresh ryegrass-based diet. Six mature fistulated sheep (30 DIM; 78.2 ± 13.53 kg) were assigned to treatments: SH (soybean hulls) and CG (corn grain), 3 ewes in each treatment in a crossover design with 14 d of adaptation. Daily ration was offered at 4% of BW in a 50:50 forage to concentrate ratio and balanced with soybean meal to achieve 16% CP (NRC, 2001). Fifty-milliliter ruminal samples were collected at 08:00, 12:00, 16:00, 20:00, 24:00 and 04:00 h and pooled per animal in each experimental period. Then a 50 mL subsample of the pooled sample (n = 18) was taken for further fat extraction and FA methylation for gas chromatography. Data were analyzed as a crossover design (Infostat, 2009), and means were compared by Tukey test (a = 0.05). No differences were observed for C8:0 C12:0, C13:1, C14:0, C14:1,C16:0, C16:2, C16:3, C18:0,C18:1t9, C18:1c11, C20:5 and C22:6 (P > 0.05) although C15:0, C16:1c9 and C17:0 were higher in SH (P < 0.05). Vaccenic acid and C18:1c9 concentrations were higher in CG than in SH (4.83 vs. 2.93; 0.81 vs. 0.38g/100g FA; P < 0.05) in contrast with linolenic acid, C18:4 and C20:4 (1.26 vs.1.79, 0.02 vs. 0.07 and 0.43 vs. 0.57g/100g FA). Surprisingly, and in contrast to several reports in cows, linoleic and rumenic acid contents tended to be higher in CG (4.21 vs.2.66 and 0.15 vs. 0.04g/100g FA) and C18:2t10c12, higher in SH (0.14 vs. 0.09 g/100g FA) (P < 0.1). Despite this fact, we have previously informed no differences in either C18:2c9t11 or C18:2t10c12 in milk fat (Miccoli et al., 2017). As less information is available in small ruminants compared with cows, results should be taken with caution. These findings are consistent with previous reports showing that concentrates alter BH pathways in a different manner among species.