The regrouping of ruminants is a common practice in dairy farms and is targeting at the improvement of milk production efficiency. However, changing the established hierarchy in a group affects both productivity and behavioral attributes. The aim of the present study was therefore to examine the possible effects of regrouping on milk yield, composition and several behavioral indices in dairy ewes. The experimental period was divided into two sub-periods of 20 days each. During the first period, 30 Chios ewes were used, while 15 Chios and 15 Karagouniko ewes were mixed during the second period. Milk yield, composition, oxidative stability, flow rate and number of kick responses during milking were recorded for each ewe. An isolation—flight distance test was also performed on a weekly basis. As indicated, the parameters recorded during the behavioral tests, such as the number of bleats, heart rate and flight distance, and the milk oxidation rate and number of kick responses were significantly increased immediately after regrouping compared to the following days during both sub-periods (p < 0.05). Moreover, milk yield was reduced in the second sub-period by 8.61% (p < 0.05). It can be concluded that regrouping could negatively influence productive and behavioral traits, and the effect was more pronounced when sheep of different breeds were mixed.