The aim of this study was to identify milk fatty acids that could indicate ketosis in early lactation cows. A total of 694 Holstein cows (314 primiparous and 380 multiparous), with average body condition score (BCS) 3.29 ± 0.62, calving from September, 2015 to August, 2016 were evaluated from a commercial dairy farm located in Palmeira county, Paraná State, Southern Brazil. A milk sample was collected from each cow between d 5 and 15 after calving, and they were frozen and stored in bottles at −20°C. Later, these milk samples, 90 from primiparous and 92 from multiparous, and one-third from healthy and two-thirds from ketotic cows, were analyzed by gas chromatography to determine 52 milk fatty acids (Finnigan Focus CG, Thermo Fisher Scientific). On d 5 and 10 after calving, BHB was also measured using a blood drop in ketone test strips (FreeStyle Optium Ketone Monitoring System, Abbott). Each association between milk fatty acid and ketosis was analyzed to obtain AUC, using MedCalc software. Averages for BHB were 1.03 ± 0.75 and 1.14 ± 0.90 mmol/L on d 5 and 10, respectively. Cows categorized as subclinical ketotic (BHB ≥1.2 mmol/L) were 26% (d 5) and 32% (d 10). Averages for milk fat and milk protein contents from the 182 samples were 4.73 ± 1.03% and 3.50 ± 0.39%, respectively, with 8.5 DIM. Milk fatty acids with AUC ≥0.90 were C12:0, C14:0, C18:1 cis 9, C17:0/C15:0, C18:1 cis 9/C14:0 ratio, and C18:1 cis 9/C15:0 ratio. The sensitivity and specificity of these variables were 90.5 and 79.2, 95.2 and 71.4, 85.7 and 84.4, 85.7 and 81.8, 91.4 and 83.1, 94.3 and 72.7, respectively. The cut-point for these fatty acids and their ratios associated with subclinical ketosis were ≤1.481, ≤6.597, >27.423, >1.359, >4.305, and >43.330 g/100 g, respectively. These results show that many fatty acids and their ratios could be used to identify ketosis in early lactation. Acknowledgment: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.