Genetic diversity studies using 39 simple-sequence repeat (SSR) markers were carried out with 114 taxa of Hydrangea macrophylla (Thunb.) Ser., including 87 H. macrophylla ssp. macrophylla cultivars and 20 members of H. macrophylla ssp. serrata (Thunb.) Makino. The SSR loci were highly variable among the taxa, producing a mean of 8.26 alleles per locus. Overall allelic richness was relatively high at 5.12 alleles per locus. H. macrophylla ssp. serrata contained nearly twice the allelic diversity of H. macrophylla ssp. macrophylla. The majority of genetic diversity was found to reside within the subspecies, with only 12% of the total genetic diversity observed occurring between subspecies. Although the elevation of H. macrophylla ssp. serrata to species level has recently been recommended by several hydrangea authorities, these data support the subspecies designation. Four cultivars (Preziosa, Pink Beauty, Tokyo Delight, and Blue Deckle) appeared to be hybrids between the two subspecies. Genetic similarities were found among five remontant cultivars (Bailmer, Oak Hill, David Ramsey, Decatur Blue, and Penny Mac) and several nonremontant cultivars, including General Vicomtesse de Vibraye, Nikko Blue, All Summer Beauty, and La France. No close genetic relationship was found between the remontant cultivar Early Sensation and other remontant cultivars. Genetic similarities were found among variegated and double-flower cultivars. Within H. macrophylla ssp. macrophylla, cultivars with mophead inflorescences clustered separately from most lacecap cultivars. This indicates the cultivars with lacecap inflorescences that were among some of the earliest introductions to Europe were not widely used in the breeding of mophead forms. Some presumed synonyms were found to be valid (‘Preziosa’ and ‘Pink Beauty’, ‘Rosalba’ and ‘Benigaku’, ‘Geoffrey Chadbund’ and ‘Mowe’), whereas others were not (‘Harlequin’ and ‘Monrey’, ‘Nigra’ and ‘Mandschurica’). This study identified potentially unexploited sources of germplasm within H. macrophylla and relationships between existing cultivars of this popular shrub. This information should be of value when selecting parents for breeding programs.