Using the nullisomic back-cross procedure, four wheat-rye chromosome substitution 2R (2D) lines with different agronomic performance, designated WR02-145-1, WR01-145-2, WR02-145-3, and WR02-145-4, were produced from a cross between 2D nullisomic wheat (Triticum aestivum L. cv. "Xiaoyan 6") and rye (Secale cereale L. cv. "German White"). The chromosomal constitution of 2n=42=21 in WR02-145 lines was confirmed by cytological and molecular cytogenetic methods. Using genomic in situ hybridization on root tip chromosome preparations, a pair of intact rye chromosomes was detected in the WR02-145 lines. PCR using chromosome-specific primers confirmed the presence of 2R chromosomes of rye in these wheat-rye lines, indicating that WR02-145 lines are disomic chromosome substitution lines 2R (2D). The WR02-145 lines are resistant to the powdery mildew (Erysiphe graminis DC. f. sp. tritici E. Marchal) isolates prevalent in northern China and may possess gene(s) for resistance to powdery mildew, which differ from the previously identified Pm7 gene located on chromosome 2RL. The newly developed "Xiaoyan 6"- "German White"2R (2D) chromosome substitution lines are genetically stable, show desirable agronomic traits, and are expected to be useful in wheat improvement.