Spatial and quantitative accumulation of the eight forms has been shown to vary during barley seed development, with tissue-specific form distribution, and relative form ratios shifting throughout early kernel formation. Kinetics of tocochromanol forms suggests specific roles throughout seed development and germination, requiring precise control of individual genes. In this study, major loci affected multiple forms, reflecting activity of genes at key intersections of the tocochromanol biosynthetic pathway. The barley 6H QTL influencing cT and dT corresponded to a rice region containing VTE4, which converts c to a and d to b, accounting for the forms mapped to this locus. Minimal FA sequence variation was observed within the VTE4 gene and its upstream regulatory region, although observed polymorphism affected significant regions, modifying two promoter motifs and causing an amino acid change. Based on VTE4 sequences of two FA RILs with divergent levels of c and d forms, it does not appear that the intronic indel plays a role in VTE4 activity. However, the indel is heritable, evidenced by the 1:1 ratio produced when genotyping the FA population with the molecular marker based on this polymorphism. Despite the sequence similarities, expression patterns of VTE4 differed between Falcon and Azhul, with cold temperatures inducing increased expression primarily in Falcon shoot tissue and Azhul embryo tissue. Since promoter sequence polymorphism affected regions involved in low-temperature response, these motifs may affect spatial expression of VTE4. Future work should be performed to explore this possibility, as well as to study additional factors affecting VTE4 gene expression such as methylation patterns and involvement of microRNA. The QTL accounting for a majority of the phenotypic variance in this study were detected on chromosome 7H. Both regions influence accumulation of multiple tocotrienol forms, the more abundant tocochromanol in barley and some other small grains. The QTL affecting all four T3 forms corresponded to the rice chromosome 6 region containing VTE2, a gene reported to preferentially add the phytyldiphosphate side chain to form tocopherols, with analogous formation of tocotrienols by HGGT. Dicots appear to lack a prenyltransferase recognizing geranylgeranyl diphosphate, hence they produce little or no tocotrienols. Monocots, however, appear to express two distinct prenyltransferases, one favoring phytyldiphosphate as substrate, the other favoring genanylgeranyl diphosphate. In this study, a major QTL on chromsosome 7H corresponded with increased accumulation of all four tocotrienol forms. Barley SNP sequences within this QTL are syntenous with a rice chromosome 6 region containing VTE2, closely neighboring a region containing HGGT. In this study, barley HGGT corresponded with the rice region containing VTE2, and a barley VTE2 locus was not identified, suggesting minimal activity of this enzyme.