The objective of this study was to identify and characterize potential biomarkers for disease resistance in bovine milk that can be used to indicate dairy cows at risk to develop future health problems. We selected high- and low-resistant cows i.e. cows that were less or more prone to develop diseases according to farmers' experience and notifications in the disease registration data. The protein composition of milk serum samples of these high- and low-resistant cows were compared using NanoLC-MS/MS. In total 78 proteins were identified and quantified of which 13 were significantly more abundant in low-resistant cows than high-resistant cows. Quantification of one of these proteins, lactoferrin (LF), by ELISA in a new and much larger set of full fat milk samples confirmed higher LF levels in low- versus high-resistant cows. These high- and low-resistant cows were selected based on comprehensive disease registration and milk recording data, and absence of disease for at least 4 weeks. Relating the experienced diseases to LF levels in milk showed that lameness was associated with higher LF levels in milk. Analysis of the prognostic value of LF showed that low-resistant cows with higher LF levels in milk had a higher risk of being culled within one year after testing than high-resistant cows. In conclusion, LF in milk are higher in low-resistant cows, are associated with lameness and may be a prognostic marker for risk of premature culling.