The aim of this paper was to evaluate the effect of negative energy balance (NEB) on milk composition including selected fatty acids of milk fat. A total of 369 dairy cows from two herds were included in the experiment and blood and milk samples were collected repeatedly. The first collection was performed in the first 35 days of lactation and the second collection between 36 and 63 days of lactation. On the basis of laboratory blood tests, dairy cows were divided into NEB+ (cows with NEB) and NEB0 (cows without NEB) groups. In total, 21% of cows had NEB. The greatest differences in milk composition were observed during the first 35 days of lactation. During this period, the NEB+ group cows had significantly higher fat content and fat/protein ratio, higher C18:0 and C18:1 content, and lower C14:0 and C16:0 content compared to the NEB0 group. These results confirm the significant effect of NEB on milk composition in early lactating dairy cows