Cotton is an economically important crop with multiple uses as fiber, biofuel, food, and feed, and drought is one of the most limiting factors in cotton production. In this study, Gossypium hirsutum ‘Acala 1517-99’, G. barbadense ‘PHY 76 Pima’ and their cross-breeding line ‘Q1735-4’ were grown in a greenhouse to characterize their growth and physiological responses to four substrate volumetric water contents (VWC) of 15, 25, 35, and 45%. An automated irrigation system consisting of soil moisture sensors, datalogger, and a relay controller was used. The results from two tests consistently showed that as VWC decreased, leaf area, stem diameter, and total dry weight (DW) decreased linearly for PHY 76 Pima and Q1735-4, but quadratically for Acala 1517-99. However, the reduction in the growth parameters varied among genotypes. As VWC was decreased from 45% to 25%, Acala 1517-99, PHY 76 Pima, and Q1735-4 exhibited reduction in height by 39.2, 32.5, and 23.7%; in leaf area by 70.9, 65.8, and 34.7%; in stem diameter by 33.4, 28.1, and 22.1%; and in total DW by 59.2, 55.6, and 15.1%, respectively. The interspecific cross-breeding line consistently displayed better drought tolerance than its parents. When VWC was further decreased to 15%, the interspecific cross-breeding line still had the lowest reduction in reproductive growth as measured by the total dry fruit weight; but its vegetative growth parameters such as plant height, leaf area, stem diameter, and total DW were similar to Acala 1517-99 and lower than PHY 76 Pima.