This experiment was conducted to study the effect of a prolonged hot period on the fatty acid (FA) composition in blood serum of dairy cows. Eighteen multiparous Holstein cows were randomly assigned to the hyperthermia group (HYP, n = 8) in August (summer season) and the control group (CON, n = 10) in October (autumn season). Blood from animals of the HYP group was collected in one heat wave, which was preceded by a long period of heat stress (HS, temperature-humidity index (THI ≥ 72)). Blood from cows of the CON group was collected under thermal comfort conditions (THI < 68). The spectrum of free fatty acids (FFA) in the blood serum was analyzed by gas chromatography. The concentration of FFA increased, including saturated FAs and monounsaturated FAs, in the blood serum of cows under conditions of prolonged HS. This was associated with the mobilization of FA into the bloodstream from adipose tissue, as a consequence of negative energy balance. An increase in the ratio of n-6/n-3 polyunsaturated FAs may indicate biomembrane dysfunction and adversely affect dairy cows. This study showed that prolonged periods of heat can affect the FA composition of blood. How much this leads to changes in the FA composition of milk and the quality of food products remains to be seen in further research.