The objective of this study was to determine whether concentrations of specific fatty acids in milk fat are a candidate for the early detection of subclinical ketosis. The case study included multiparous cows fed a lipogenic diet or a mixed glucogenic:lipogenic diet during the first 9 wk of lactation. Milk fatty acid profiles of cows classified as healthy (n = 8) or as subclinically ketotic (n = 8) based on a blood plasma beta-hydroxybutyrate threshold concentration of 1.2 mmol/L were compared. Subclinically ketotic cows showed an elevated proportion of C18:1 cis-9 in milk fat during the whole registration period.