The aim of this study was to correlate serum β-hydroxybutyrate (BHB) concentrations with milk fatty acids in early lactation cows. Six hundred and 80 Holstein cows (306 primiparous and 374 multiparous) calving from September, 2015 to August, 2016 were evaluated in a commercial dairy farm located in Palmeira county, Paraná State, Southern Brazil. A milk sample was collected from each cow between the 5th and the 15th day after calving, and they were frozen and stored in bottles at −20°C. Later, 90 (44 from primiparous and 46 from multiparous) samples were analyzed by gas chromatography to determine 52 fatty acids profile (Finnigan Focus CG, Thermo Fisher Scientific). On the same day of milk sampling, a blood sample was collected to analyze serum BHB in automatic biochemical analyzer (BS-200, Mindray, Shenzhen, China). On d 5 (D5) and 10 (D10) after calving, BHBA was also measured using a blood drop in ketone test strips (FreeStyle Optium Ketone Monitoring System, Abbot). Averages for milk fat and milk protein percentages were 4.56 ± 1.12% and 3.56 ± 0.43%, respectively, with 9.2 DIM. Averages for BHBA were 1.03 ± 0.75 and 1.14 ± 0.90 mmol/L on D5 and D10, respectively. Cows categorized as subclinical ketotic (BHB ≥1.2 mmol/L) were 26% (D5) and 32% (D10). Moderate negative correlations were detected between BHB and C4:0, C6:0, C8:0, C10:0, C10:1, C11:0, C12:0, C13:0 iso, C13:0, C14:0, C15:0 iso, C15:0 anteiso, C15:0 and C15:0 anteiso/C17:0 ratio (r = −0.49, −0.61, −0.56, −0.55, −0.43, −0.45, −0.52, −0.57, −0.53, −0.56, −0.44, −0.47, −0.51, and −0.51, respectively), showing that cows with intense negative energy balance (NEB) have impaired de novo fatty acids synthesis in mammary gland. Moderate positive correlations were observed between BHB and C18:1 cis-9, C18:1 cis-11, C18:1 cis-12, C18:1 cis-13 and C16:1 cis-9/C15:0 ratio (r = 0.51, 0.51, 0.51, 0.43, and 0.54, respectively), being that C18:1 cis-9 has been suggested in other studies as an important biomarker for NEB. In summary, several fatty acids and their ratios can be used to identify postpartum dairy cows with intense NEB.