We studied tree water uptake patterns, tested for complementary water use among species and analysed controlling factors in a tropical tree diversity experiment. The water uptake depth of five species was investigated across seasons and diversity levels using the natural abundance of water isotopes (δ2H, δ18O) and modelling. Three distinct water acquisition strategies were found for trees growing in monocultures during the dry season: strong reliance (>70%) on soil water from the upper layer (0–30 cm) (Cedrela odorata), uptake from the upper and deeper layers (>30 cm) in equal proportions (Hura crepitans, Anacardium excelsum and Luehea seemannii) and water uptake predominately from deeper layers (Tabebuia rosea). Seasonal shifts in water uptake were most pronounced for T. rosea. The water uptake pattern of a given species was independent of the diversity level underlining the importance of species identity and species characteristics in spatial and temporal tree water use. Statistics did not show a significant effect of diversity on source water fractions, but we did see some evidence for complementary water resource utilization in mixed species plots, especially in the dry season. Our results also demonstrated that the depth of soil water uptake was related to leaf phenology and tree transpiration rates. A higher proportion of water obtained from deeper soil layers was associated with a high percentage foliage cover in the dry season, which explained the higher transpiration rates.