Many important plant species have polyploidy in their recent ancestry, complicating inferences about the genetic basis of trait variation. Although the principal locus controlling the proportion of polyunsaturated fatty acids (PUFAs) in seeds of Arabidopsis thaliana is known (fatty acid desaturase 2; FAD2), commercial cultivars of a related crop, oilseed rape (Brassica napus), with very low PUFA content have yet to be developed. We showed that a cultivar of oilseed rape with lower than usual PUFA content has non-functional alleles at three of the four orthologous FAD2 loci. To explore the genetic basis further, we developed an ethyl methanesulphonate mutagenised population, JBnaCAB_E, and used it to identify lines that also carried mutations in the remaining functional copy. This confirmed the hypothesised basis of variation, resulting in an allelic series of mutant lines showing a spectrum of PUFA contents of seed oil. Several lines had PUFA content of ~6 % and oleic acid content of ~84 %, achieving a long-standing industry objective: very high oleic, very low PUFA rapeseed without the use of genetic modification technology. The population contains a high rate of mutations and represents an important resource for research in B. napus.Electronic supplementary materialThe online version of this article (doi:10.1007/s11032-013-9954-5) contains supplementary material, which is available to authorized users.