Radiation hybrid (RH) mapping is based on radiation-induced chromosome breakage and analysis of chromosome segment retention or loss using molecular markers. In durum wheat (Triticum turgidum L., AABB), an alloplasmic durum line [(lo) durum] has been identified with chromosome 1D of T. aestivum L. (AABBDD) carrying the species cytoplasm-specific (scsae) gene. The chromosome 1D of this line segregates as a whole without recombination, precluding the use of conventional genome mapping. A radiation hybrid mapping population was developed from a hemizygous (lo) scsae− line using 35 krad gamma rays. The analysis of 87 individuals of this population with 39 molecular markers mapped on chromosome 1D revealed 88 radiation-induced breaks in this chromosome. This number of chromosome 1D breaks is eight times higher than the number of previously identified breaks and should result in a 10-fold increase in mapping resolution compared to what was previously possible. The analysis of molecular marker retention in our radiation hybrid mapping panel allowed the localization of scsae and 8 linked markers on the long arm of chromosome 1D. This constitutes the first report of using RH mapping to localize a gene in wheat and illustrates that this approach is feasible in a species with a large complex genome.