The stem rust (Puccinia graminis Pers.:Pers. f.sp. tritici Eriks. and Henn.) resistance gene Sr39, which confers resistance to TTKSK (Ug99), has been incorporated into the wheat (Triticum aestivum L.) genome from Aegil-ops speltoides in the form of a chromosome translocation but it has not been deployed into adapted cultivars. In this study, we characterized translocation lines carrying Sr39 in four different wheat backgrounds with fluorescent genomic in situ hybridization (GISH) and simple sequence repeat (SSR) markers. The results indicated that RL5711 and RL6082 had translo-cation chromosomes of comparable structure. The translocation chromosome in PI 600683 has lost Ae. speltoides chromatin in the telo-meric end of the 2S long arm. Six translocation lines derived from the cross PI 600683/3*HY438 had translocation chromosomes of comparable structure to the one found in PI 600683. However, one line (P9714-AM03C51), showed a substantial reduction in Ae. speltoides chromatin in the short arm of the translocated chromosome. The study demonstrated that it is apparently feasible to shorten Ae. speltoides chromatin in some wheat-Ae. speltoides translocation lines. These results and the identification of diagnostic SSR markers will be useful in guiding chromosome manipulation efforts to further shorten the Ae. speltoides chromosome segments in these materials. Greenhouse inoculation of translocation lines with stem rust indicated that the Sr39 gene conditions resistance to at least seven stem rust races.