Excessive levels of boron (B) in the subsoil can severely limit crop yields, particularly under dryland conditions where crops often have to depend on rainfall and available water stored in the soil during fallow. In bread wheat (Triticum aestivum L.), boron tolerance is controlled by at least two loci located on chromosomes 4AL (Bo4) and 7BL (Bo1). In this study, we sought to determine whether novel genomic regions can be identified in wheat primary synthetic hexaploids (SHWs). The study used data from 333 SHW lines imported into Australia in different shipments between 2003 and 2007. On average, the SHWs were uniformly more tolerant to boron toxicity than the sensitive check, Meering, and the top 5 % showed tolerance levels that were superior (P ≤ 0.05) to that of Halberd, the most tolerant wheat check cultivar. At a genome-wide significance threshold of −log(P) ≥ 2.8, association analyses using different algorithms consistently identified three DArT markers, two on chromosome 1AL and one on 4AL. The 4AL region was localised close to the deletion bin location of Bo4, the root-specific boron transporter gene. On the other hand, the two loci on chromosome 1A represent novel regions, which when validated will increase the options of achieving tolerance beyond that conferred by Bo1 and Bo4 alone in breeding programmes.