Habitat loss and fragmentation due to urbanization are the most pervasive threats to biodiversity in southern California. Loss of habitat and fragmentation can lower migration rates and genetic connectivity among remaining populations of native species, reducing genetic variability and increasing extinction risk. However, it may be difficult to separate the effects of recent anthropogenic fragmentation from the genetic signature of prehistoric fragmentation due to natural geologic and climatic changes. We examined the phylogenetic and population genetic structure of a flightless insect endemic to cismontane southern California , Stenopelmatus “mahogani” (Orthoptera: Stenopelmatidae). We combined population genetic analyses of mtDNA sequence data with GIS-based reconstructions of current and prehistoric habitat fragmentation to test whether patterns of differentiation correspond to current and/or prehistoric levels of habitat fragmentation. Our results suggest that recent fragmentation due to urbanization has measurably increased genetic differentiation beyond the effects of prehistoric fragmentation. Simulations confirm that contemporary patterns of genetic structure could be produced by recent urban fragmentation using biologically reasonable assumptions about model parameters. In addition, diversity within populations was positively correlated with fragment size, suggesting that increased drift in smaller fragments has lowered genetic variability. Loss of genetic connectivity and diversity can hinder a population’s ability to adapt to ecological perturbations commonly associated with urbanization, such as habitat degradation, climatic changes and introduced species. Consequently, our results underscore the importance of preserving and restoring landscape connectivity for long-term persistence of low vagility native species.