The decline of eastern box turtle populations is associated with habitat loss and fragmentation, lack of recruitment into breeding populations, removal of individuals from the wild for pets, and road mortality. Box turtle populations in many areas of the eastern United States may effectively be isolated as high traffic volumes on roads adjacent to turtle habitats prevent successful dispersal. If so, populations surrounded by heavily used roads may be less genetically diverse than those in relatively intact habitats. I investigated whether populations in three Montgomery County, Maryland parks that were surrounded by roads were genetically differentiated relative to populations in two larger habitats in Maryland's Prince George's and Anne Arundel counties. Sampling was conducted between 2005 and 2008. I used 10 microsatellite markers to compare these populations, and sampled in an additional five sites (two in Maryland, and one each in Pennsylvania, the District of Columbia, and Florida), to better assess population structure. I found little evidence of genetic differentiation among central Maryland populations regardless of the extent of isolation. I attribute these findings to the slow rate of change in turtle evolution; the observed similarities in genetic diversity may reflect past rather than present gene flow. I found moderate to great differentiation in populations separated by substantial distances. To determine whether road mortality exceeds additive mortality levels believed to be a threat to population persistence, I estimated population size in the three Montgomery County, Maryland, parks using mark-recapture techniques, and compared these estimates to the number of dead, injured, and live turtles in or very near roads observed during walking and driving surveys conducted in 2006. Road-based morality rates fell within the range estimated to be inconsistent with population growth in one of the parks. Road mortality appeared to affect females out of proportion to their abundance in the population. Suggestions for reducing box turtle mortality in areas associated with high mortality rates are included.