Biofumigation refers to the suppression of soil-borne pests and pathogens by biocidal compounds released in soil when glucosinolates (GSL) in Brassica green manure or rotation crops are hydrolysed. We investigated the potential to enhance biofumigation by considering the variation in GSL production in the roots and shoots of 76 entries from 13 Brassica and related weed species grown in the field. Total plant GSL production on a ground area basis at mid-flowering ranged from 0.8 to 45.3 mmol m−2. The variation derived equally from differences in biomass and GSL concentration, which were not correlated in either root or shoot tissues. Roots (0–0.15 m) contributed an average of 23.6% (range 2–81%) of the total plant GSLs, their contribution limited by low biomass rather than GSL concentration, which was usually similar or higher than that of shoots. The GSL concentrations in root and shoot tissues did not correlate significantly with seed levels in any of the species, so selection for higher plant GSL production to enhance biofumigation potential cannot be based on seed GSL levels. The types of GSLs present in the tissues varied considerably between species but were consistent within species. In contrast, the concentration of individual and total GSLs in both root and shoot tissues varied four to ten-fold both between and within all species. Shoots contained predominately aliphatic GSLs, while aromatic GSLs, particularly 2-phenylethyl GSL, were dominant in the roots of all entries. Indolyl GSLs were present in all tissues but at low concentrations (<1 μmol g−1). The variation in the biomass, GSL profiles and concentrations in both roots and shoots provide significant scope to select or develop brassicas with enhanced biofumigation potential. Further studies on the efficacy of the various GSL hydrolysis products to suppress target organisms in soil are required to fully exploit biofumigation as a part of integrated pest management.