The San Bernardino flying squirrel (Glaucomys oregonensis californicus) is thought to be the southernmost population of the Humboldt's flying squirrel (G. oregonensis) and is restricted to the San Bernardino and San Jacinto Mountains in California. However, recent surveys indicate they have been extirpated from the latter locality. In this study, we characterized the intraspecific genetic structure and diversity of G. oregonensis. We amplified and sequenced at least 300 bp of the mitochondrial cytochrome-b gene and 11 nuclear microsatellites to provide baseline genetic data for this species and subspecies, investigate G. oregonensis genetic structure with a focus on the subspecies distributed in California, and finally evaluate the genetic diversity within G. o. californicus. Population and phylogenetic analyses were performed on a total of 147 samples (tissue, hair, and museum specimen) using the Illumina high-throughput sequencing (HTS) platform, thereby bioinformatically coding microsatellite alleles using established pipelines. Our results indicate reduced genetic diversity in G. o. californicus and highlight genetic distinctiveness of the San Jacinto population. In addition, the differences in genetic diversity between the mitochondrial and nuclear markers suggest sex-biased dispersal and historic separation of the species in multiple refugia followed by more recent gene flow among subpopulations in northern California. This study contributes valuable information toward understanding the genetic variation within G. oregonensis, provides information for future conservation decisions for G. o. californicus, and has novel implications for HTS genotyping-by-sequencing.