Insufficient dietary intake of micronutrients contributes to the onset of deficiencies termed hidden hunger-a global health problem affecting approximately 2 billion people. Vitamin B₁(thiamine) and vitamin B₆(pyridoxine) are essential micronutrients because of their roles as enzymatic cofactors in all organisms. Metabolic engineering attempts to biofortify rice endosperm-a poor source of several micronutrients leading to deficiencies when consumed monotonously-have led to only minimal improvements in vitamin B₁and B₆contents. To determine if rice germplasm could be exploited for biofortification of rice endosperm, we screened 59 genetically diverse accessions under greenhouse conditions for variation in vitamin B₁and vitamin B₆contents across three tissue types (leaves, unpolished and polished grain). Accessions from low, intermediate and high vitamin categories that had similar vitamin levels in two greenhouse experiments were chosen for in-depth vitamer profiling and selected biosynthesis gene expression analyses. Vitamin B₁and B₆contents in polished seeds varied almost 4-fold. Genes encoding select vitamin B₁and B₆biosynthesisde novoenzymes (THICfor vitamin B₁,PDX1.3a-candPDX2for vitamin B₆) were differentially expressed in leaves across accessions contrasting in their respective vitamin contents. These expression levels did not correlate with leaf and unpolished seed vitamin contents, except forTHICexpression in leaves that was positively correlated with total vitamin B₁contents in polished seeds. This study expands our knowledge of diversity in micronutrient traits in rice germplasm and provides insights into the expression of genes for vitamin B₁and B₆biosynthesis in rice.