The objective of this study was to evaluate the effect of goat diet supplementation with fish oil on the profile of milk fatty acids, particularly the polyunsaturated fatty acids (PUFA). The benefits of increased levels of PUFA in the diet of humans are numerous. A total of 10 Alpine goats were randomly selected from the dairy herd of the International Goat Research Center at Prairie View A&M University. The animals were divided into 2 groups (5 animals/group) and each group was assigned to either the control or experimental diet. The control animals received the basal diet without supplementation of fish oil, whereas the experimental group was given the same basal diet with 1.14 g of fish oil/kg of concentrate. The total feeding duration of this experiment was 8 weeks with 2 weeks of the same basal diet as the adaptation period. Milk samples were collected and analyzed for chemical composition at wk 0, 2, 4, and 6. The fatty acid profile of collected milk samples were analyzed using GC-MS. The data were analyzed using the PROC GLM procedure of SAS based on repeated measure design. The model included diet and time as the main effects and their interaction. All tests to determine statistical differences were at 5% significance level. Compared with the control group, the level of fish oil supplementation did not affect the total fat content of goat milk with diet as the main effect. However, the protein and SNF contents of milk from the experimental group were significantly higher (P < 0.05) than the milk of the control group. With respect to time as the main effect, significant differences (P < 0.05) were observed in the quantities of the following fatty acids: C6:0, C8:0, C10:0, C12:0, C14:0, C14:1, C15:0, C15:1, CLA trans-10,cis-12, C22:0, and C20:4?-6 during the study period. Based on the results obtained, the level of fish oil supplementation in the diet used in this study did not affect the total fatty acids content of goat milk, suggesting that higher levels of fish oil may be required to increase the fatty acid profile of goat milk.