Habitat fragmentation is a leading threat to wildlife globally. Effects of habitat fragmentation are often inferred retrospectively based on genetic connectivity estimated from a single modern sampling effort. Alternatively, genetic data obtained from museum specimens can facilitate direct comparisons of genetic connectivity pre- and post-habitat fragmentation. We examined the effects of habitat fragmentation on the federally endangered salt marsh harvest mouse endemic to the San Francisco Bay Area by comparing genetic data from 11 microsatellite markers in modern samples (2010-2022) and historical museum specimens (1938-1959). Genetic diversity (allelic richness, expected heterozygosity) was greater in the historical samples in the total population and within each of the six subpopulations we sampled. Genetic distance (FST) among subpopulations was greater in the modern sample than historical sample, consistent with reduced connectivity and/or genetic drift within populations. Generally, sites with the smallest remaining habitat patches exhibited the greatest degree of genetic diversity loss. Our results directly link losses in genetic diversity and connectivity with ~80 years of habitat loss and fragmentation. Our work highlights the threat of anthropogenic landscape change to wildlife populations.