ABSTRACT: Sperm whales (Physeter macrocephalus) resident to the Gulf of Mexico are geographically isolated from other populations, making them vulnerable to the impacts of oil and gas exploration prevalent in the region. Despite long-standing research focused on sperm whales in the Gulf of Mexico, genetic structure within this population has not yet been investigated, nor an assessment of the impacts of the 2010 Deepwater Horizon oil spill on population structure. Here, we investigated the spatial structure and temporal variation in mitochondrial DNA (mtDNA) haplotype frequencies from 619 genetic samples collected from 377 individual sperm whales identified by microsatellite genotyping. This study uses samples collected over nearly two decades from 2000-2017 to support previous findings of genetic isolation of the Gulf of Mexico from populations in the Western North Atlantic (FST = 0.242, p < 0.001) and Caribbean (FST = 0.423, p < 0.001). In addition, we demonstrate significant changes in population structure within the Gulf of Mexico across both space and time. Most notably, the Central subregion, where the 2010 Deepwater Horizon oil spill originated, underwent a significant change in haplotype frequencies across time (FST = 0.141, p < 0.001), with implications for disruption of matrilineal population structure. Our findings of spatial and temporal structure within Gulf of Mexico sperm whales highlights the importance of regular monitoring and the establishment of baseline population data in the face of anthropogenic and environmental impacts.