The distribution of Y-chromosomal variation from the 12 Y-SNP and 17 Y-STR markers was determined in six major provinces (Seoul-Gyeonggi, Gangwon, Chungcheong, Jeolla, Gyeongsang, and Jeju) to evaluate these populations' possible genetic structure and differentiation in Korea. As part of the present study, a 10-plex SNaPshot assay and two singleplex SNaPshot assays were developed. Based on the result of 12 Y-SNP markers (M9, M45, M89, M119, M122, M174, M175, M214, RPS4Y, P31, SRY465, and 47z), almost 78.9% of tested samples belonged to haplogroup O-M175 (including its subhaplogroups O3-M122: 44.3%, O2b*-SRY465: 22.5%, O2b1-47z: 8.7%), and 12.6% of the tested samples belonged to haplogroup C-RPS4Y. A total of 475 haplotypes were identified using 17 Y-STR markers included in the Yfiler kit, among which 452 (95.2%) were individual-specific. The overall haplotype diversity for the 17 Y-STR loci was 0.9997 and the discrimination capacity was 0.9387. Pairwise genetic distances and AMOVA of the studied Korean provinces reflected no patrilineal substructure in Korea, except for Jeju Island. Thus, this survey shows that the present data of Korean individuals could be helpful to establish a comprehensive forensic reference database for frequency estimation.