Malnutrition is a widespread human health concern in regions where diets rely heavily on a few staple crops that lack sufficient micronutrient levels. Biofortification, the genetic improvement of nutrient content in crops, has become a key goal of plant breeding. Vitamin B3 (niacin: nicotinamide and nicotinic acid) is an essential nutrient largely unavailable in maize-based diets, primarily due to trigonelline, a methylated plant-active derivative of nicotinic acid. Trigonelline makes up a major portion of niacin-related compounds in maize grain but does not release bioavailable nicotinic acid during digestion. Identifying the genes involved in niacin accumulation and its conversion to trigonelline could improve the nutritional value of maize for human consumption. To explore this, we analysed niacin and trigonelline levels in grain from ~1,700 maize inbred lines and identified three lines with near-zero trigonelline and approximately 50% higher niacin levels. One of these extreme lines was crossed with a maize inbred line having average levels of both traits to construct a biparental population of ~250 F2 individuals for genetic mapping of these metabolites in grain. This analysis identified a major locus that explained a large portion of the variation in trigonelline concentration. Among the genes in this interval, a potential causal gene—a methyltransferase—had a ~5 kb transposon inserted in its first exon in the extreme parental inbred line. A genetic marker for the presence or absence of the transposon insertion perfectly cosegregated with the trigonelline phenotype in the F2 population. Ongoing work aims to confirm the role of the methyltransferase and further uncover genetic regulation of niacin and trigonelline accumulation, with the aim of guiding future maize breeding efforts to reduce niacin deficiencies globally.