Coicis oil is one of the main functional substances of Coix seed, and fatty acid is an important component. The contents of stearic acid, oleic acid and linoleic acid in seed kernels of 190 Coix germplasms from 9 provinces in China were detected. ClSAD and ClFAD2 sequences polymorphisms were analyzed and the haplotypes were identified. The haplotype association analysis of fatty acid content was carried out. The results showed that there were wide variations in the content of three fatty acids in the seeds of different Coix germplasms, among which the content of oleic acid was the highest and the content of stearic acid was the lowest, and there was a very significant positive correlation between the fatty acid components. There were 14 SNPs and 3 SNPs in ClSAD and ClFAD2, and 5 haplotypes were identified respectively. Hap1 and Hap3 of ClSAD were conducive to the conversion of stearic acid to oleic acid, Hap1 of ClFAD2 was conducive to the accumulation of linoleic acid, and one key SNP locus was identified in each of the two genes. The results will provide a theoretical basis for the breeding of high oil Coix varieties, as well as the development of molecular markers and the analysis of related molecular mechanisms.