Water availability is one of the main limitations of potato yields due to the high sensitivity of this crop to water deficit. The objective of this study was to determine the effect of water deficit on some physiological and biochemical responses in yellow diploid potato plants (Solanum tuberosum L. Group Phureja) of the cultivars Criolla Colombia, Criolla Dorada and Criolla Ocarina. Plants at tuber initiation were subjected to two treatments: continuous irrigation and water deficit imposed by withholding water at tuber initiation for 17 d. The results showed that plants under water deficit increased chlorophyll concentration, malondialdehyde and proline content. However, these plants showed a decrease in stomatal conductance, leafarea, total dry mass and exhibited a higher root/shoot ratio in all potato cultivars. In addition, all the cultivars also showed a decrease in yield, which was associated with sensitivity to water stress. Although the high content of proline and high root/shoot ratio may be associated with tolerance to water deficit, this association was not observed in these cultivars, probably due to the high reduction of stomatal conductance, which limited the production of photoassimilates, plant growth, and,therefore, the yield.