Hydrothermal grown ZnO single crystals were annealed in N2 or O2 between 900 and 1300 °C. Positron lifetime measurements reveal a single lifetime in all the ZnO samples before and after annealing. The positron lifetime is about 181 ps after annealing at 900 °C in either N2 or O2 atmosphere. However, increase of the positron lifetime is observed after further annealing the sample at higher temperatures up to 1300 °C, and it has a faster increase in O2 ambient. Temperature dependence measurements show that the positron lifetime has very slight increase with temperature for the 900 °C annealed sample, while it shows notable variation for the sample annealed at 1300 °C. This implied that annealing at high temperature introduces additional defects. These defects are supposed to be Zn vacancy-related defects. Cathodoluminescence (CL) measurements indicates enhancement of both UV and green emission after annealing, and the enhancement of green emission is much stronger for the samples annealed in O2 ambient. The possible origin of green emission is tentatively discussed.