Stable isotopic composition of groundwaters and their hydrochemistry have been used for investigating possible existence of both palaeo-water and replenishable groundwater in Pushkar (Ajmer) Valley, Rajasthan. Causes of salinity increase in some of the groundwaters and intermixing of saline and fresh groundwaters have also been studied. The delta O-18 values of water samples show a wide range varying from -6.3 parts per thousand to +4.0 parts per thousand, indicating different sources of origin of these waters. There is a depletion of delta O-18 with increasing depth of water table which may be due to stratification in groundwater with shallower zones being recharged from relatively modern rainfall. The chemical composition of groundwaters shows a wide variation with electrical conductivity ranging from 150 mumhos/cm to 8400 mumhos/cm. Linear relationship between delta O-18 and Cl- is a clear indication that the groundwaters are freshwater intermixed with different amounts of a saline endmember. Highly depleted, low salinity groundwaters seem to be of palaeo-origin. The most probable genesis of the O-18 enriched, high salinity groundwater is from an originally evaporitic environment.