NIR spectroscopy making use of a near infrared spectrum offers wide using in a food quality control. This quick method allows the measuring of numerous samples with minimal usage of chemicals. Due this fact it is also used in quantitative analyses of milk composition. In this study we observed an impact of different temperatures of cow’s milk samples on measuring exactness. The milk samples were taken from separate dairy cows and immediately refrigerated to 6–8 °C. Until the analysis the samples were deposited in the refrigerator. Just before the analysis on FT NIR spectrometer they were warmed to 40 °C and then cooled down in water bath to the temperatures 20 °C, 21 °C, 23 °C and 25 °C. Afterwards the samples were measured on Petri dish in reflectance mode with resolution 8 and number of scans 100. The spectres of samples were evaluated using calibration models for determination of solids and contents of proteins, fat and lactose. Observed measures were statistically compared within separate temperatures in relation to standard temperature 20 °C.