AbstractThe Masai giraffe has experienced a population decline from 70,000 to 35,000 in the past three decades and was declared an endangered subspecies by the IUCN in 2019. The remaining number of Masai giraffe are geographically separated by the steep cliffs of the Gregory Rift escarpments (GRE) in Tanzania and Kenya dividing them into two populations, one west and one east of the GRE. The cliffs of the GRE are formidable barriers to east–west dispersal and gene flow and the few remaining natural corridors through the GRE are occupied by human settlements. To assess the impact of the GRE on Masai giraffe gene flow, we examined whole genome sequences of nuclear and mitochondrial DNA (mtDNA) variation in populations located east (Tarangire ecosystem) and west (Serengeti ecosystem) of the GRE in northern Tanzania. Evidence from mtDNA variation, which measures female‐mediated gene flow, suggests that females have not migrated across the GRE between populations in the Serengeti and Tarangire ecosystems in the past ~289,000 years. The analysis of nuclear DNA variation compared to mtDNA DNA variation suggests that male‐mediated gene flow across the GRE has occurred more recently but stopped a few thousand years ago. Our findings show that Masai giraffes are split into two populations and fulfill the criteria for designation as distinct evolutionary significant units (ESUs), which we denote as western Masai giraffe and eastern Masai giraffe. While establishing giraffe dispersal corridors across the GRE is impractical, conservation efforts should be focused on maintaining connectivity among populations within each of these two populations. The importance of these efforts is heightened by our finding that the inbreeding coefficients are high in some of these Masai giraffe populations, which could result in inbreeding depression in the small and fragmented populations.