Understanding the genetic variation of functional components in Yunnan barley landrace will benefit the genetic improvement and developing health food. In total, 236 accession Yunnan barley landraces were used to compare the difference of resistant starch(RS), total flavones(FL), alkaloids(AL) and γ-aminobutyric acid(GABA) of barley grains. The results showed that significant difference in RS, FL, AL and GABA contents existed among 249 barley accessions. Comparisons of RS and FL contents of different barley types showed that the difference between multi-row and two row, purple grain and yellow grain barley was significant at 0.01 level, respectively. Meanwhile, significant difference of AL and GABA contents also existed between multi-row and two row, hull and hulless barley, respectively. 236 Yunnan barley landraces could be clustered into 10 clusters. The fourth cluster and eighth cluster are outstanding. Distances of final cluster centers is furthest between first cluster and seventh cluster, and that is nearest between first cluster and tenth cluster. It would be advantageous to the choice of breeding material. Twenty accessions with good functional components were selected for possible parents to improve productivity of barley in Yunnan.