Lentils are a nutritious food in the human diet. High in protein and with low glycemic index, lentils are also a source of folate and other B vitamins. Understanding variability in B vitamin contents among lentils will allow breeders to select for increased levels. We analyzed 34 cultivated and three wild genotypes for vitamins B1, B2, B3, B5, B6, B7, and B9 in the cotyledons and seed coats. Variation for all B vitamins was observed across the genotypes. Cotyledons had higher concentrations of B1 and B3, while seed coats had higher concentrations of B2, B5, B6, and B9. Wild accessions had the highest concentrations of vitamin B9 and were among the highest for B2. Differential distribution of B vitamins across seed tissues and lentil genotypes has implications for consumption and for breeding. There is useful genetic variability which could be used to increase B vitamin levels in future lentil varieties.