The aim of this study was to investigate the effect pre-partum habituation in the milking parlour on behaviour and lactation performance of buffalo heifers. Sixteen buffalo heifers in late gestation were used for this study. The animals, with an age ranging from 30 to 44 months at the start of the study, were equally allocated into two treatments and balanced for estimated calving date. Eight animals received pre-partum habituation for 10 days before the estimated calving date (group H), while the eight others received no treatment and served as the control (group C). During the pre-partum habituation sessions, group H animals were moved to the milking parlour and left for 10 min in the milking stall once each day, where the udder was thoroughly washed with warm water, wiped with a disposable towel and massaged. The behaviours, registered from entrance into the milking stall to exit, were step and kick. After calving milk flow profiles, milk yield and milk quality variables were determined along with behavioural recordings. The H animals during the habituation procedure showed a reduction in the number of steps (P < 0.001) and kicks (P < 0.01). After calving the H animals performed fewer steps than the control animals at 0 (P < 0.001), 3 (P < 0.01), 6 (P < 0.01), 13 (P < 0.01) and 20 (P < 0.01) days after calving. Animals from group H also performed fewer kicks than control animals at 0 (P < 0.001), 3 (P < 0.01), 6 (P < 0.01) and 13 (P < 0.01) days after calving. In the C group a reduction in the number of steps (P < 0.001) and kicks (P < 0.001) was observed as lactation proceeded, whereas for group H only a tendency for a reduction over lactation was detected. Pre-partum habituation did not significantly affect milk quality or milk flow variables. Milk yield in the first 3 min of milking (P < 0.001), and average milk flow (P < 0.001), increased throughout the experimental period, whereas the duration of the pre-milking phase decreased as lactation proceeded (P < 0.001). This study shows that buffalo heifers exposed to a pre-partum habituation programme performed fewer steps and kicks than control animals during milking. Therefore, it is concluded that using this treatment can reduce the level of restlessness in buffalo heifers during milking.