Three chestnut species (Castanea sativa, Castanea crenata and Castanea mollissima) and their hybrids have been massively planted worldwide for nut production. To better study and manage these trees, we identified nuclear single nucleotide polymorphism (SNP) markers using restriction-associated DNA sequencing. Out of 343 loci tested using the MassARRAY platform, 237 were successfully typed. Two sets of multiplexed SNP markers were then selected that include 79 polymorphic markers, 68 of which withhold stringent quality criteria such as quasi-systematic amplification across species and Mendelian segregation in both purebred and hybrid individuals. They provide sufficient power for species, hybrids and backcross characterization as well as for clonal identification, as shown by a comparison with a dataset including 94 single sequenced repeat (SSR) loci.