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Single-nucleotide polymorphisms (SNPs) are the most commonly used DNA markers in population genetic studies. We used the Illumina HiSeq4000 platform to develop single-nucleotide polymorphism (SNP) markers for Manila clam <i>Ruditapes philippinarum</i> using restriction site-associated DNA sequencing (RAD-seq) genotyping. Eighty-eight SNP markers were successfully developed by using high-resolution melting (HRM) analysis, with a success rate of 44%. SNP markers were analyzed for genetic diversity in two clam populations. The observed heterozygosity per locus ranged from 0 to 0.9515, while the expected heterozygosity per locus ranged from 0.0629 to 0.4997. The value of F_IS was estimated to be from −0.9643 to 1.0000. The global F_st value was 0.1248 (<i>p</i> < 0.001). After Bonferroni correction, 15 loci deviated significantly from the Hardy–Weinberg equilibrium (<i>p</i> < 0.0006). These SNP markers provide a valuable resource for population and conservation genetics studies in this commercially important species.