Micronutrients and vitamins are chemical elements required in trace quantities for normal human growth and development. Micronutrients and vitamin deficiency is prevalent throughout the world. The first objective of this research was to determine folate concentration in 10 lentil genotypes and evaluate the effect of environment on folate concentration. Folate concentration ranged from 216 to 290 μg/100 g with a mean of 255 μg/100 g and the concentration differed across years and locations. A significant genotype × environment interaction effect was observed for lentil folate concentration. The second objective was to measure the iron, zinc, copper, calcium and magnesium concentration in 26 cultivated and wild lentils. Significant variation in Fe, Zn, Cu, Ca, and Mg concentration among Lens species and no single genotype had high concentrations of all micronutrients.The third objective was to determine genetic diversity among 29 cultivated and wild lentils using 39 simple sequence repeat markers. Thirteen of 39 SSR markers were polymorphic among the 29 lentil genotypes. Cluster analysis grouped the genotypes into 4 clusters broadly based on the genotyping data and this grouping had correspondence with the pedigree relationships of the genotypes. The fourth objective was to develop expressed sequence tags-simple sequence repeats (EST-SSRs) markers in lentil. Lentil EST sequences (9513) from the NCBI database were assembled into 4053 unigenes. Unigenes were screened for simple sequence repeats and 348 primer pairs were designed. Fifty-seven primer pairs were polymorphic among the 22 lentil genotypes providing additional gene-specific primers for use in lentil breeding. The fifth objective was to develop gene specific molecular markers for iron metabolism related genes in lentil and to study their gene expression in the presence of excess iron. Gene specific markers were developed for Ferritin-1, BHLH-1, and IRT-1 to allow detailed study of the iron metabolic pathway in lentil. Differential gene expression of Ferritin-1 and IRT-1 under excess iron was observed at 2 hours but not at 8 hours and 24 hours. Results of these studies contribute to a broad understanding of the genetic variation, environmental influence on and expression of genes related to micronutrient and vitamin concentration and metabolism in lentil.