Glucosinolates (GSLs) are sulphur- and nitrogen-containing secondary metabolites implicated in the fitness of Brassicaceae and appreciated for their pungency and health-conferring properties. In Indian mustard ( L.), GSL content and composition are seed-quality-determining traits affecting its economic value. Depending on the end use, i.e., condiment or oil, different GSL levels constitute breeding targets. The genetic control of GSL accumulation in Indian mustard, however, is poorly understood, and current knowledge of GSL biosynthesis and regulation is largely based on . A genome-wide association study was carried out to dissect the genetic architecture of total GSL content and the content of two major GSLs, sinigrin and gluconapin, in a diverse panel of 158 Indian mustard lines, which broadly grouped into a South Asia cluster and outside-South-Asia cluster. Using 14,125 single-nucleotide polymorphisms (SNPs) as genotyping input, seven distinct significant associations were discovered for total GSL content, eight associations for sinigrin content and 19 for gluconapin. Close homologues of known GSL structural and regulatory genes were identified as candidate genes in proximity to peak SNPs. Our results provide a comprehensive map of the genetic control of GLS biosynthesis in Indian mustard, including priority targets for further investigation and molecular marker development.