Marine turtle species consist of several genetically discrete ‘evolutionarily significant units’ (ESUs) which do not interbreed. We studied Flatback turtle (Natator depressus) hatchlings from two rookeries (Mon Repos Conservation Park and Bare Sand Island, Australia) representing two separate ESUs. Turtles from these ESUs differ in several key life history traits, including body size, and we predicted hatchlings would also differ in locomotor performance. We also investigated the proportion of hatchlings with non-modal scute patterns to determine whether this varies between ESUs. We collected newly emerged hatchlings, and measured mass, carapace length and width, and recorded the scute pattern. We then measured self-righting ability and crawling speed. Our results confirmed a difference in hatchling size between the two ESUs, with Mon Repos rookery hatchlings being larger. However the size difference did not translate into a difference in self-righting ability or crawling speed. The Mon Repos rookery also produced a larger proportion of hatchlings with major non-modal scute pattern compared to Bare Sand Island rookery. The differences suggest hatchling survival rates may differ between ESUs, and that ESUs should be studied separately when implementing conservation measures.