Abstract The Colorado River Basin (CRB) is experiencing persistent aridification due to a complex interplay of natural and anthropogenic activities, resulting in significant groundwater depletion across the region. We used over two decades of NASA GRACE and GRACE Follow‐On (GRACE‐FO) observations (April 2002–October 2024), land surface models and observed data, to document pronounced groundwater depletion in the CRB. We estimate that the CRB lost 52.2 ± 4.0 km3 of terrestrial water storage over the study period, of which groundwater accounted for 65% (34.3 ± 9.2 km3). Of this, the Upper Basin lost 14.6 ± 3.5 km3 of terrestrial water storage (53% from groundwater, 7.8 ± 5.3 km3) while the Lower Basin lost 36.0 ± 6.2 km3 of terrestrial water storage (71% from groundwater, 25.5 ± 7.4 km3). Progress toward groundwater sustainability could be achieved by reducing annual extraction in line with the annual depletion rates presented here (0.35 km3/yr in Upper Basin and 1.15 km3/yr in the Lower Basin).