Rhododendron canescens is a deciduous azalea native to the southeastern United States that is used in landscaping due to its ornamental qualities. A genotyping-by-sequencing (GBS) approach was taken to characterize the genetic structure and diversity of a R. canescens germplasm collection. Single nucleotide polymorphisms (SNPs) were identified by two software platforms, STACKS and GBS-SNP-CROP. Three distinct R. canescens populations were detected by STRUCTURE analysis with GBS-SNP-CROP data, whereas two populations were distinguished using STACKS data. Principal component analysis (PCA) with data from both SNP pipelines supported the presence of three populations. Statistical results indicated that there was low genetic differentiation between the populations, but relatively high genetic diversity within populations. The inbreeding coefficient of the R. canescens accessions was low, which would be expected with an outcrossing species. These results suggest that there may be a significant level of gene flow between populations of R. canescens.