Human activities often induce maladaptive effects in urban wildlife, such as increased inbreeding and decreased gene flow, consequently leading to a loss of biodiversity. Although maladaptive effects are commonly reported in urban wildlife, some wildlife species are not affected. Understanding the conditions and factors that affect the genetic structure of wildlife is crucial for its conservation in urban environments. Eurasian red squirrels Sciurus vulgaris in Obihiro City, Hokkaido, Japan, have settled in large residential lots with a high risk of vehicle collisions for approximately 30 years, raising the possibility that the urban population would be genetically isolated from nearby rural populations and may experience a rapid decrease in genetic diversity. In this study, we analysed the population structure, genetic distance, and nucleotide diversity of squirrels in the Obihiro area using genome-wide single nucleotide polymorphisms (SNPs). Principal component analysis and ADMIXTURE showed that urban and rural squirrel populations formed distinct clusters with intermediate urban and rural boundary populations. Genetic distances between the urban populations were greater than that of other pairs. Nucleotide diversities were lower in urban populations than in other populations, suggesting limited gene flow between urban and other populations, possibly due to urbanisation. The change in the genetic characteristics of urban squirrel populations during this short period demonstrates the strong impact of urbanisation. Our findings suggest that a genome-wide approach to the genetic structure of wildlife contributes to its conservation in urban environments.