Occurrence of uranium (U) in the groundwater of south-west Punjab is a matter of great concern due to the rise of contaminant levels at an alarming rate, its spatial and vertical variability and poor understanding of uranium mobility with depth. Present study has been conducted to investigate the vertical extent of contamination and geochemical factors controlling U mobilization in the groundwater of semi-arid Punjab. Out of total 140 samples collected and analyzed, 76% samples have uranium levels greater than the chemical toxicity limit of World Health Organization (WHO, 30 μg.L−1) and 34% samples have concentration higher than the radiological toxicity limit given by Atomic Energy Regulatory Board (AERB, 60 μg.L−1). Water at shallow depth (<60 m) is found to be more contaminated than water at deeper depth (>60 m) and concentration lies in the range of 4.26–318.03 μg.L−1. Spatial distribution graphs are used to identify the hotspots of contamination and concentration is higher in the flow direction of groundwater towards south-west. In both the aquifers, groundwater is alkaline and oxic in character with carbonate weathering as dominant hydrogeochemical process affecting chemical composition of water. Higher uranium concentration is seen in the oxidizing, alkaline and carbonate-rich water. Alkali earth metals (Na+ & K+) are also strongly correlated with the uranium in groundwater at both the levels. Use of environmental stable isotopes (δ18O & δ2H) shows the evaporation signature in both the aquifers. Health risk assessment is carried out and values higher than permissible limit give indication towards the health risk to the population exposed due to the consumption of uranium contaminated water from long time. This study provides a strong base for better understanding the source of uranium in the aquifer system of region and the results would be useful for further studies in quaternary alluvial aquifers of semi-arid regions.