Metabolic disorders in early lactation have negative effects on dairy cow health and farm profitability. One method for monitoring the metabolic status of cows is metabolic profiling, which uses associations between the concentrations of several metabolites in serum and the presence of metabolic disorders. In this cross-sectional study, we investigated the use of mid-infrared (MIR) spectroscopy of milk for predicting the concentrations of these metabolites in serum. Between July and October 2017, serum samples were taken from 773 early-lactation Holstein Friesian cows located on 4 farms in the Gippsland region of southeastern Victoria, Australia, on the same day as milk recording. The concentrations in sera of β-hydroxybutyrate (BHB), fatty acids, urea, Ca, Mg, albumin, and globulins were measured by a commercial diagnostic laboratory. Optimal concentration ranges for each of the 7 metabolites were obtained from the literature. Animals were classified as being either affected or unaffected with metabolic disturbances based on these ranges. Milk samples were analyzed by MIR spectroscopy. The relationships between serum metabolite concentrations and MIR spectra were investigated using partial least squares regression. Partial least squares discriminant analyses (PLS-DA) were used to classify animals as being affected or not affected with metabolic disorders. Calibration equations were constructed using data from a randomly selected subset of cows (n = 579). Data from the remaining cows (n = 194) were used for validation. The coefficient of determination (R2) of serum BHB, fatty acids, and urea predictions were 0.48, 0.61, and 0.90, respectively. Predictions of Ca, Mg, albumin, and globulin concentrations were poor (0.06 ≤ R2 ≤ 0.17). The PLS-DA models could predict elevated fatty acid and urea concentrations with an accuracy of approximately 77 and 94%, respectively. A second independent validation data set was assembled in March 2018, comprising blood and milk samples taken from 105 autumn-calving cows of various breeds. The accuracies of BHB and fatty acid predictions were similar to those obtained using the first validation data set. The PLS-DA results were difficult to interpret due to the low prevalence of metabolic disorders in the data set. Our results demonstrate that MIR spectroscopy of milk shows promise for predicting the concentration of BHB, fatty acids, and urea in serum; however, more data are needed to improve prediction accuracies.