Arabinoxylans (AX) are prebiotics found naturally in wheat (Triticum aestivum L.) flour with well known beneficial effects on human health. Arabinoxylan concentration was measured in wheat grain of the Berkut × Krichauffdoubled haploid (DH) population grown at two contrasting environments in South Australia; one at an adequaterainfall site in Roseworthy in 2009 and one at a low-rainfall site in Minnipa in 2007. A linkage map of 528 genetic markers was used for quantitative trait loci (QTL) mapping. There was wide variation in the grain AX concentrations within the DH population in both environments, ranging from 5.4 to 8.5% (of dry weight), and there was a significant phenotypic correlation between two environments. Heritability was estimated as 0.51. Quantitative trait loci associated with grain AX concentrations were located on chromosomes 1A, 2A, 3D, 4D, 6B, and 7A. Quantitative trait loci on 2A (QGax.aww-2A.1) and 4D (QGax.aww- 4D.1) had major effects. At QGax.aww-2A.1, the favorable allele came from Berkut, while at QGax.aww-4D.1, the favorable allele was derived from Krichauff. Effects of markers at these two QTLs were further validated using grain from more environments (Roseworthy, Minnipa, and Booleroo, South Australia, in 2006). In all cases, lines carrying both favorable alleles at those loci contributed a significant increase in wheat grain AX concentration compared to lines without the favorable alleles. These genome regions could therefore be useful targets for wheat breeding or mapping of candidate genes controlling grain AX accumulation.