Targeted genotyping of transcriptome-scale genetic markers is highly attractive for genetic， ecological， and evolutionary studies， but achieving this goal in a cost-effective manner remains a major challenge， especially for laboratories working on nonmodel organisms. Here， we develop a high-throughput， sequencing-based GoldenGate approach (called HD-Marker)， which addresses the array-related issues of original GoldenGate methodology and allows for highly multiplexed and flexible targeted genotyping of more than 12，000 loci in a single-tube assay (in contrast to fewer than 3100 in the original GoldenGate assay). We perform extensive analyses to demonstrate the power and performance of HD-Marker on various multiplex levels (296， 795， 1293， and 12，472 genic SNPs) across two sequencing platforms in two nonmodel species (the scallops and )， with extremely high capture rate (98%-99%) and genotyping accuracy (97%-99%). We also demonstrate the potential of HD-Marker for high-throughput targeted genotyping of alternative marker types (e.g.， microsatellites and indels). With its remarkable cost-effectiveness (as low as $0.002 per genotype) and high flexibility in choice of multiplex levels and marker types， HD-Marker provides a highly attractive tool over array-based platforms for fulfilling genome/transcriptome-wide targeted genotyping applications， especially in nonmodel organisms.