We hypothesized that alterations in metabolic status systematically affect the milk fatty acid (FA) composition and thus milk FA can reveal the metabolic status of dairy cows. We, therefore, investigated changes in the milk FA profile of 24 Simmental cows fed high-quality ryegrass hay (HQ hay) rich in water-soluble carbohydrates (WSC) in the diet, which was shown to affect energy partitioning towards either milk production or body reserve retention, and consequently the metabolic status depending on the dietary proportion of concentrates supplied. The test diets (per kg dry matter (DM)) were CON (600 g fiber-rich hay + 400 g concentrate), 60HQ (600 g HQ hay + 400 g concentrate), 75HQ (750 g HQ hay + 250 g concentrate), and 100HQ (100 g HQ hay). As opposed to the fiber-rich hay, the HQ hay was richer in crude protein (CP; 223 vs. 65 g/kg CP in DM), WSC (198 vs. 110 g/kg WSC in DM) and 18:3 n-3 (560 vs. 316 g/kg FA). The feeding trial was performed from 10 d before calving until 28 d thereafter and milk samples were collected on 7, 14, 21 and 28 d after calving for the FA analysis. Linear and non-linear regression analyses of milk FA and serum metabolites (NEFA and BHB) concentrations were performed. Energy secreted with the milk relative to the intake was highest in cows fed CON and 100HQ and lowest in the 75HQ cows. Both energy-deficient groups (CON and 100HQ) showed elevated levels of milk 18:1 n-9 at the expense of some mammary de novo FA compared to the other two groups (P < 0.05). Cows fed 60HQ secreted greater amounts (g/d) of the de novo FA compared to 75HQ and CON (P < 0.05). The HQ hay promoted the level of conjugated linoleic acids in milk regardless of the inclusion level, and, when used at 75 % or more in the diet, also promoted the level of 18:3 n-3 (P < 0.001) due to increased 18:3 n-3 intake with the hay. The modeled data using the ratios of 15:0 to 18:1 n-9, 14:0 to 18:1 n-9 and the sum of de novo FA to 18:1 n-9 were consistently associated with serum concentrations of NEFA and BHB with a ratio below 0.02, 0.2 and 0.4 indicating excessive mobilization and greater risk of metabolic disturbances. The relationships between the FA ratios and BHB were confirmed with a meta-analysis using means of published studies. In summary, milk FA reflect the metabolic status of early lactation dairy cows. Feeding HQ hay generally promotes health beneficial C18 FA in milk.