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The Gharb aquifer plays a critical role as a source of drinking water and irrigation in Morocco. However, rapid economic growth and increased use of chemical fertilizers have led to groundwater pollution and land degradation. To address this issue, a vulnerability assessment was conducted using the Susceptibility Index (SI) method. Five parameters including depth to groundwater, effective recharge, slope, soil type, and land use/land cover were considered to evaluate groundwater vulnerability. The vulnerability assessment revealed index values ranging from 31 to 160. The study area was categorized into three zones of relative vulnerability (low, moderate, and high). Spatial analysis demonstrated significant spatial heterogeneity, with moderate vulnerability observed in areas characterized by shallow groundwater and composed primarily of sandstones in the northwest and southwest regions. In the entire study area, the low, medium, and high vulnerability zones covered 3%, 70%, and 26% (101 km2, 2725 km2, and 986 km2), respectively. Furthermore, Tritium isotope analysis and nitrate content were selected and applied as pollution markers to confirm and validate the obtained vulnerability map. The Tritium (3H) content in groundwater reveals a correlation with an R2 value of 0.86 with the degree of vulnerability, while the nitrate concentration in the aquifer’s groundwater showed a correlation with an R2 value of 0.55.