Dairy cows in negative energy balance (NEB) must compensate for the lack of energy by lipomobilisation, which is manifested by the release of large amounts of fatty acids (FA) into the blood as non-esterified FA (NEFA). These are then incorporated directly into milk fat. The aim of this study was to investigate the relationships between serum NEFA levels and milk FA contents and to determine the strength of these relationships in dairy cows with different degrees of NEB. Eight blood and milk samples were collected from each of 66 dairy cows during the first 8 weeks of lactation. Blood serum was analysed for NEFA levels to determine the risk of NEB, while FA contents were measured from milk samples. Cows were then divided into 3 groups according to serum NEFA concentrations within 14 days postpartum: without NEB (NEB0; NEFA < 0.6 mmol/l), with NEB (NEB1; once NEFA > 0.6 mmol/l) and with deep NEB (NEB2; twice NEFA > 0.6 mmol/l). The correlations between NEFA and milk FAs were calculated and the differences between the groups of dairy cows were evaluated. Moderate correlations were observed between serum NEFA and long-chain FA (LCFA) (r = 0.56), monounsaturated FA (MUFA) (r = 0.47), C16:0 (r = -0.58), C18:0 (r = 0.44) and C18:1 (r = 0.50) in all dairy cows regardless of NEB group. The correlations between NEFA and milk parameters in the NEB0 and NEB1 groups were weak to moderate. In the NEB2 group, however, the correlations were stronger, with correlation coefficients for C16:0 and LCFA being r = -0.65 and r = 0.63, respectively. Least squares means were calculated for all the parameters determined in the milk from different cow groups. The differences were statistically significant (P < 0.05) between the NEB0 and NEB2 groups for the same parameters that correlated with NEFA, i.e. C16:0, C18:0, C18:1, LCFA and MUFA. The results show that the correlations between NEFA and MK in milk increased as the state of NEB became more severe. In conclusion, concentrations of some milk fatty acids may play a significant role in predicting cows with deeper NEB (i.e., with more frequent occurrence of above-limit NEFA values (NEFA GREATER-THAN OR EQUAL TO 0.6 mmol/l) in the first 2 weeks of lactation) and thus at higher risk of NEB-related diseases.