Sorghum seed is traditionally used as secondary food sources in addition to rice in Korea. While the hypoglycemia regulating phytochemicals have been found in sorghum seed, peptides related with hypoglycemia never been studied before. To obtain the peptide characteristics and the specifically high-expressed peptides in hypoglycemic sorghum seed, peptide profiles of seven hypoglycemic and five non-hypoglycemic sorghum lines bred in RDA were determined using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). The twelve sorghum lines exhibited 104 peptides on CM10 protein chip array (weak cation exchange) and 95 peptides on Q10 (weak cation exchange) in the molecular mass range from 2,000 to 20,000 Da. Heat map via supervised hierarchical clustering of the significantly different peptides (p < 0.01) in peak intensity among the 12 lines effectively revealed the specifically upregulated peptides in each line and distinguished between 7 hypoglycemic and 5 non-hypoglycemic lines. Through the comparison with hypoglycemic and non-hypoglycemic lines, 10 peptides including 2231.6, 2845.4, 2907.9, 3063.5, 3132.6, 3520.8, 4078.8, 5066.2, 5296.5, 5375.5 Da were specifically high-expressed in hypoglycemic lines at p < 0.00001. This study characterized seed peptides of 12 sorghums and found ten peptides highly expressed for hypoglycemic sorghum lines, which could be used as peptide biomarkers for identification of hypoglycemic sorghum.