The aim of this study was to prove the influence of different ways of sheep milking on the concentration of Cortisol in blood serum. Twenty crossbreed Šumavská sheep and Lacaune (SxLA) and twenty East Friesian Sheep (EF) were divided into two groups. First group (ten ewes) was milked by hand and the other group (ten ewes) was milked by machine during two lactation period. Blood samples were taken during evening milking at the beginning and at the end of lactation. The experimental data were analyzed by a General Linear Model from the StatSoft statistical package. In machine and hand milked crossbreed (S x LA) the concentrations of Cortisol were significantly lower (P < 0.01) before milking than concentration of Cortisol measured after milking. There was not proved statistically significant difference in Cortisol concentrations between hand milked sheep group and machine milked sheep group.