Boron (B) is an essential micronutrient required for optimal plant growth and development. Seed yield and quality are often compromised in plants grown under limited soil B. Indian mustard (Brassica juncea L.) is one of the most susceptible plants to B limitation. In this study, twenty-seven Indian mustard genotypes were grown in hydroponic culture with different B concentrations, and growth performances were evaluated based on their root and shoot growth. Geeta, RH406, and Maya were found to be low-B tolerant, moderate, and sensitive genotypes, respectively. In Geeta, root meristem cells showed higher viability than RH406 and Maya under low B conditions. Geeta had a significantly higher B concentration in the root and youngest opened leaf (YOL) than Maya and RH406. The pectin levels in the cell wall of the root and the YOL varied across different genotypes under different B conditions. The pectin levels in the cell wall of YOL were lowest in Geeta and highest in Maya under sufficient B conditions. Based on these results, we suggest that a higher B uptake and translocation and possibly lower pectin levels are the key factors responsible for low B tolerance in B. juncea genotypes.