Patterns of genetic variation within a species may be a consequence of historical factors, such as past fragmentation, as well as current barriers to gene flow. Using sequence data from the mitochondrial cytochrome oxidase subunit II region (COII) and the nuclear gene wingless, we conducted a phylogeographical study of the holarctic skipper Hesperia comma to elucidate patterns of genetic diversity and to infer historical and contemporary processes maintaining genetic variation. One hundred and fifty-one individuals were sampled from throughout North America and Eurasia, focusing on California and adjacent regions in the western United States where morphological diversity is highest compared to the rest of the range. Analyses of sequence data obtained from both genes revealed a well-supported division between the Old and New World. Within western North America, wingless shows little geographical structure, while a hierarchical analysis of genetic diversity of COII sequences indicates three major clades: a western clade in Oregon and Northern California, an eastern clade including the Great Basin, Rocky Mountains and British Columbia, and a third clade in southern California. The Sierra Nevada and the Transverse Ranges appear to be the major barriers to gene flow for H. comma in the western United States. Relatively reduced haplotype diversity in Eurasia compared to North America suggests that populations on the two continents have been affected by different historical processes.