The underlying mechanisms of the triple-oxygen (O-16, O-17, and O-18) isotopic content of deuterated (D) isotopologues of water in H-D exchange reactions in the gas phase remain elusive. Herein, we have demonstrated a highresolution gas-phase spectral analysis of doubly (D2O) and singly (HDO) deuterated isotopologues of water in the region around 7.8 ym using quantum cascade laser-based cavity ring-down spectroscopy. Isotopic fractionations among doubly and singly deuterated species of water, (D2O)-O-16, (HDO)-O-16, (HDO)-O-17, and (HDO)-O-18, in the gas phase were carried out by probing the fundamental and hot band transitions in the nu(2) (bending) mode of D2O and the fundamental nu(2) transitions for the other water isotopes. We subsequently investigated the fractionations of different D-enriched water isotopologues for the H-D exchange reaction using various mixtures of D2O in H2O. We explored the potential role of triple-oxygen isotopic contents through enrichments and depletions of HD16O, HD17O, and HD18O, involved in the H-D reaction. Our first clear, direct, and quantitative experimental evidence reveals a new picture of gas-phase isotopic fractionation chemistry in a mixture of light and heavy water (H2O-D2O).