Hydrangea macrophylla (bigleaf hydrangea) is one of the most important floral and nursery crops worldwide. However, breeding of new bigleaf hydrangea cultivars has been hampered by a long breeding cycle and lack of genetic resources. This study investigated the genetic diversity and population structure of 82 bigleaf hydrangea cultivars using single-nucleotide polymorphisms (SNPs) originated from genotyping-by-sequencing. A total of 5803 high-quality SNPs were discovered in a bigleaf hydrangea cultivar panel. A phylogenetic analysis and analysis of molecular variance based on discovered SNPs concluded the taxonomic classification of H. macrophylla ssp. serrata as a subspecies of H. macrophylla. Principal component analysis confirmed ‘Preziosa’ as a hybrid between H. macrophylla ssp. macrophylla and H. macrophylla ssp. serrata. In addition, the cultivar Lady in Red was also found to be a hybrid between the two subspecies. The population structure analysis identified three groups among the 82 cultivars. All H. macrophylla ssp. serrata cultivars belonged to one group, and two groups were revealed within H. macrophylla ssp. macrophylla. The separation within H. macrophylla ssp. macrophylla indicated a second gene pool due to breeding efforts that have targeted similar breeding goals for bigleaf hydrangea. The discovered SNPs and the phylogenetic results will facilitate further exploitation and understanding of phylogenetic relationships of bigleaf hydrangea and will serve as a reference for hydrangea breeding improvements.