Definition and establishment of a fixed reference lactation length could provide useful tools for on-farm comparison of ewes and flock management as well as genetic evaluations for the breeding programme. The objectives of this study were to (i) evaluate different reference lactation lengths for the Chios dairy sheep and (ii) define the most suitable reference length for the breed. A total of 260 042 test-day milk records from 24 474 ewes in 130 flocks collected between 2003 and 2014 were used; 15 different lactation lengths were evaluated ranging from 120 to 260 days, defined at 10-day intervals as reference for the Chios sheep. The evaluation criteria included: (a) heritability and repeatability of milk yield in each reference lactation, (b) genetic correlation of reference lactation milk yield with actual lactation milk yield and yield at first test-day record and (c) correlated response in reference lactation milk yield from selection based on first test-day milk yield. The latter emulates genetic gains achieved for milk yield based on early lactation selection. Heritability and repeatability estimates of reference lactation milk yield and genetic correlation with actual lactation yield favoured long reference lactations (180 to 230 days). On the contrary, correlation with first test-day record milk yield was higher for short lactations (120 to 170 days). Moreover, selection on first test-day record milk yield would lead to a correlated response in reference yield in 220 days equal to 85% of the highest estimate achieved in the maximum reference length of 260 days (190 days when only considering first lactation milk yield). Based on the results of the present study, an overall reference lactation length for the Chios breed of 220 days post-lambing and a first lactation reference length of 190 days post-lambing are recommended.