Interspecific hybridization is a common breeding approach for introducing novel traits and genetic diversity to breeding populations. Southern highbush blueberry (SHB) is a blueberry cultivar group that has been intensively bred over the last 60 years. Specifically, it was developed by multiple interspecific crosses between northern highbush blueberry [NHB, Vaccinium corymbosum L. (2n = 4x = 48)] and low-chill Vaccinium species to expand the geographic limits of highbush blueberry production. In this study, we genotyped polyploid blueberries, including 105 SHB, 17 NHB, and 10 rabbiteye blueberry (RE) (Vaccinium virgatum Aiton), from the accessions planted at Poplarville, Mississippi, and accessions distributed in Japan, based on the double-digest restriction site-associated DNA sequencing. The genome-wide SNP data clearly indicated that RE cultivars were genetically distinct from SHB and NHB cultivars, whereas NHB and SHB were genetically indistinguishable. The population structure results appeared to reflect the differences in the allele selection strategies that breeders used for developing germplasm adapted to local climates. The genotype data implied that there are no or very few genomic segments that were commonly introgressed from low-chill Vaccinium species to the SHB genome. Principal component analysis-based outlier detection analysis found a few loci associated with a variable that could partially differentiate NHB and SHB. These SNP loci were detected in Mb-scale haplotype blocks and may be close to the functional genes related to SHB development. Collectively, the data generated in this study suggest a polygenic adaptation of SHB to the southern climate, and may be relevant for future population-scale genome-wide analyses of blueberry.