Abstract: The content of H2O spin isomers in saturated vapor during its fast evaporation from the surface of water in a cell at room temperature has been analyzed. The experiment was performed using the two-compartment scheme of simultaneous fast laser analysis of the concentration ratio of H2O ortho- and para-isomers in saturated vapor in two analytical cells. One of these cells cointained water and its saturated vapor. The other was previously evacuated, and saturated vapor from the first cell was fed to it. Simultaneous analysis of the relative content of ortho- and para-water molecules during gas probe sampling, which lasted about 0.8 s, was performed with a rate of 10 measurements per second using a two-channel spectrophotometer based on tunable diode lasers (TDLs). The spectral range near 7355 cm–1, where the ortho- and para-Н2О absorption lines are closely located, was chosen for analysis. During the fast evaporation, which accompanied saturated vapor sampling, the evaporating vapor was found to be enriched with ortho-H2O isomer, whose content amounted to ~20% of the equilibrium value 3 : 1. An important fact is that this result is indicative of possible enhanced mobility and the absence of hydrogen bonds for some of ortho-Н2О molecules in the surface water layer, which is consistent with the known fact that water vapor transmitted through porous materials becomes enriched with ortho-H2O isomers. © Allerton Press, Inc. 2024. ISSN 1541-308X, Physics of Wave Phenomena, 2024, Vol. 32, No. 3, pp. 241–248. Allerton Press, Inc., 2024.