Flax is a special oil crop cultivated in the northern China and rich on unsaturated fatty acids. Breeding of high linolenic acid varieties and high linoleic acid varieties is the main objective in flax quality breeding programs. Molecular marker assisted selection can improve flax breeding efficiency and shorten breeding duration. In this study， analysis of fatty acid phynotypic datasets gained from a natural population of 246 flax accessions， together with the construction of genotyping population， the KASP genotyping and verification of linolenic acid and linoleic acid-related SNP loci were carried out. We obtained the following results mainly including： （1） The linolenic acid was significantly negatively correlated with other components， and especially the negative correlation between linolenic acid and linoleic acid was the highest ； （2） Linolenic acid and linoleic acid-related SNP genotyping populations were established consisting of 87 accessions， which included 49 high linoleic acid （≥54%） accessions， 36 low linoleic acid （≤13.5%） accessions， and two accessions No.8 （CH-89） and NO. 254 （CI637PI91037） simultaneously showing high linoleic acid and low linolenic acid； （3） Seven SNPs associated with particular traits were validated， i.e. three SNPs g6-6229142， g10-19305239， and g9-18961021 were significantly associated with linolenic acid， four SNPs g6-19208888， g9-14900088， g15-22369840， and g2-7680441 were significantly associated with linoleic acid； （4） A coincidence of the 7 SNP loci was detected by high-throughput sequencing and KASP genotyping， confirming the effectiveness and accuracy of the KASP genotyping.