Overexploitation of large and highly connected marine fish populations may impact their future evolutionary potential and recovery, but understanding the underlying changes to genetic diversity can be challenging. The collapse of Northern Cod (Gadus morhua), historically the largest population of Atlantic Cod in the northwest Atlantic, raised questions regarding the potential biological consequences for the populations’ genetic diversity. Using low-coverage whole genome sequencing (lcWGS) on collections from the 1990s and 2010s, we detected a decline in genetic diversity of Atlantic Cod along the Canadian Atlantic coast. Most recent collections exhibited lower genetic diversity (i.e., Watterson’s θ) and fewer genetically distinguishable groups than 1990s collections. Runs of homozygosity were also longer and more numerous in the 2010s collections. Our results demonstrate a loss in genetic diversity at the population and individual level following the fishery collapse and indicate that genetic diversity can be lost even in numerically large populations. The loss of genetic diversity may have important implications for fisheries conservation.