This study aimed to evaluate the effect of inclusion of low purity glycerin in the diet on the fatty acid composition of milk of crossbred cows. We used 15 crossbred Holstein x Zebu cows distributed in three Latin squares 5 x 5, the following treatments: control (no addition of glycerin in the diet); inclusion of 5% glycerin in the diet dry matter; inclusion of 10% glycerol in the diet dry matter; inclusion of 15% glycerol in the diet dry matter, and the inclusion of 20% glycerol in the diet dry matter. The inclusion of glycerin in the diet had no effect (P<0.05) the concentration of butyric acid (C4:0). In contrast, we observed a quadratic effect (P<0.05) on the concentrations of fatty acids caproic C-6:0 (= 11.33 the point of maximum), caprylic C-8:0 (= 9.50 the point of maximum) and capric C-10:0 (= 6.66 the point of maximum) also short chain fatty acids, following the same trend of milk production. Decreased linearly (P<0.05) concentrations of medium chain fatty acids, fatty acids Lauric C-12:0, Myristic C-14:0, myristoleic C-14:1, pentadecanoic C-15:0, Pentadecenoico: C-15:1, Palmitic C-16:0 and palmitoleic C-16:1. Was observed increased linearly (P<0.05) on concentrations of polyunsaturated fatty acids and monounsaturated. In contrast, the concentration of saturated fatty acids decreased linearly (P<0.05). The concentration of fatty acids Omega 6 series showed decreasing linear effect (P<0.05). As for the concentration of Omega 3, there was no effect of adding glycerin. The PUFA SFA-1 ratio showed linear growth (P<0.05) and the ratio n-6 n-3(-1) showed a quadratic effect (P<0.05), with of 6.66 the point of maximum. The concentration of conjugated linoleic acid total had increased linearly (P<0.05). It is recommended the inclusion of glycerin in the diet of dairy cows to the level of 20% based on dry matter.