To facilitate the utility of SNP-based genotyping, we developed a new method called target SNP-seq which combines the advantages of multiplex PCR amplification and high throughput sequencing. Compared with KASP, Microarrays, GBS and other SNP genotyping methods, target SNP-seq is flexible both in SNPs and samples, yields high accuracy, especially when genotyping genome wide perfect SNPs with high polymorphism and conserved flanking sequences, and is cost-effective, requiring 3 days and $7 for per DNA sample to genotype hundreds of SNP loci. The present study established a DNA fingerprint of 261 cucumber varieties by target SNP-seq with 163 perfect SNPs from 4,612,350 SNPs based on 182 cucumber resequencing datasets. Four distinct subpopulations were found in 261 Chinese cucumber varieties: the north China type, the south China type, the Europe type, and the Xishuangbanna type. The north China type and Xishuangbanna type harbored lower genetic diversity, indicating greater risk of genetic erosion in these two subpopulations. Furthermore, a core set of 24 SNPs was able to distinguish 99% of the 261 cucumber varieties. 29 core cucumber backbone varieties in China were identified. Therefore, target SNP-seq provides a new way to screen out core SNP loci from the whole genome for DNA fingerprinting of crop varieties. The high efficiency and low cost of target SNP-seq is more competitive than the current SNP genotyping methods, and it has excellent application prospects in genetic research, as well as in promoting plant breeding processes in the near future.