Technical Abstract: In wheat (Triticum aestivum L.), stem rust resistance gene Sr39, derived from Aegilops speltoides Tausch, is highly resistant to multiple stem rust races including TTKSK (Ug99). However, the gene has not been used in wheat breeding because of linkage drag associated with the large 2S chromosome segment on which Sr39 is located in the original 2S/2B translocation stocks (RL5711 and RL6082). To make this valuable gene usable to wheat breeders, we have conducted research on reducing the size of the alien chromatin surrounding Sr39 using chromosome engineering in conjunction with stem rust testing and high-throughput molecular marker genotyping. ‘Chinese Spring’ (CS) ph1b mutant was used to cross with translocation stock RL6082 and then backcrossed to the F1 hybrids. The BC1F1 plants were screened with the stem rust pathogen and molecular markers to detect homozygous ph1b plants. Resistant BC1F1 plants homozygous for ph1b were selected and backcrossed to CS. A population of 1,048 BC2F1 plants was tested with stem rust and a co-dominant microsatellite marker. We identified 40 resistant plants with dissociation of the marker allele from the 2S chromosome segment. Four wheat lines, designated as RWG1, RWG2, RWG3, and RWG4, carrying Sr39 on minimal alien chromatins have been identified and verified by using fluorescent genomic in situ hybridization (FGISH) and by testing with eight races of stem rust. Based on the sequences of the deletion bin-mapped wheat ESTs and the collinear region of the rice (Oryza sativa) genome, we developed three co-dominant STS molecular markers tightly linked to Sr39 on the short alien chromatins. The new wheat germplasm lines and Sr39-linked STS markers are important resources for breeding commercial wheat cultivars resistant to TTKSK and other races of stem rust.