Abstract: There are variety of B vitamins in wheat grain, such as thiamine, riboflavin and pyridoxine. The objective of this study was to understand the content of B vitamins (thiamine, riboflavin and pyridoxine) in colored grain wheat, and their Patterns of variation, and provide a theoretical basis for optimization of B vitamins content in wheat breeding. A specific Ultra performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) method was developed to accurately determine content in wheat varieties with different color grains. Electrospray MS was operated in postive ion mode and monitored using multiple reaction monitoring (MRM). Spectrophotometry was used to determine the contents of pigment. The results indicated that there are variations in B vitamins content in Black Wheat 76 and VICTO and its twelve sib-lines of their hybridized offspring. The difference of B vitamins content for Dongjian series was also extremely significant. The contents of B vitamins and total anthocyanin in wheat grain did not show significant correlations. Deep colored grain of wheat was not rich in B vitamins. There are essential differences for B vitamins content of wheat grains among genotypes, controlled by genetic effects. Genetic improvement can raise the B vitamins content of wheat grain. The B vitamins contents are significantly different between the black grain wheat 76 and VICTO, and twelve Sib-lines of their hybridized offspring. Significant heterobeltiosis was achieved through sexual hybridization for breeding wheat cultivars with high B vitamins content.