The South Moravian region of Pasohlávky and the Lower Austrian town of Laa an der Thaya belongs to the areas of intensive use of thermal mineral water for recreational and balneological purposes. Thermal mineral water is extracted from a deep-seated Jurassic aquifer, mainly composed of carbonates. These carbonates overlie the crystalline bedrock, which is dipping towards the southeast below the Outer Western Carpathians. Due to this trend, there are two developments of the Jurassic sediments that differ in their lithological composition and are vertically separated by the Musov transition zone. To the northwest from this zone, there is a shallower carbonate development and in the southeast, there is a deeper development which is composed of permeable carbonates together with impermeable marls. The Neogene sediments of Carpathian foredeep are deposited on the top of the Jurassic carbonate structure. Thermal mineral water is exploited by two hydrogeological wells – Muš-3G (Pasohlávky, 1 455 m deep) and Laa Th N1 (Laa an der Thaya, 1 448 m deep) situated in the shallower carbonate part of the Jurassic aquifer. The aim of this study, that summarizes the important partial outputs of the cross-border project Interreg HTPO (Hydrothermal Potential of the Area, ATCZ167), is to specify and clarify the origin, genesis and the processes of water formation based on the hydrochemical evaluation of thermal mineral water. For this purpose, both the final reports of studied wells and especially results from newly performed analyses of stable isotopes of hydrogen ?2H and oxygen ?18O were used. The ?18O and ?2H values of water samples from the Muš-3G well varies from -12,77 ‰ to -12,03 ‰ and from -92,23 ‰ to -88,05 ‰, respectively, while water samples from the Laa Th N1 well are isotopically heavier with ?18O values ranging from -7,04 ‰ to -6,31 ‰ and with ?2H values ranging from -51,04 ‰ to -49,33 ‰. In the Pasohlávky region we suppose it is an isotopically depleted water that has infiltrated in the cold climatic period (glacial). Evaluation of the chemical composition revealed that although the thermal waters from both areas of the hydrogeological structure are of the same Na-Cl type, their total mineralization differs significantly. Lower total mineralization in the Pasohlávky area (approx. 2,2 g/L) is also associated with higher relative concentrations of bicarbonate ions (HCO3-) and atmogenic nitrogen, typically bound to infiltrated meteoric waters. The thermal water in this area is mostly of meteoric origin, mixed with primary marinogenic water. In the Laa an der Thaya region, there is water with higher total mineralization (approx. 11 g/L) and lower relative concentrations of HCO3-, which together with the results of isotope analyses indicates a higher content of primary seawater. The marinogenic origin of waters also confirms the presence of bromides and iodides. The process of mixing primary marine waters with infiltrated meteoric waters significantly contributes to the chemical composition of the studied waters in both areas, while the original marinogenic water type of Na-Cl is preserved.