Agropyron cristatum (2n = 4x = 28, PPPP) is a wild relative of common wheat and confers several desirable agronomic traits to wheat, such as high grain number per spike and enhanced resistance to certain diseases. Development of wheat – A. cristatum 6P translocation lines facilitates its utilization in wheat improvement. In this study, 26 wheat – A. cristatum 6P translocation lines were characterized by in situ hybridization (ISH) and 6P-specific sequence-tagged-site (STS) markers. These translocation lines carried different 6P chromosomal segments, which covered the whole 6P chromosome. FISH results showed that 15, 5, and 6 lines were translocated onto wheat A, B, and D genomes, respectively. Compared with the previous reports, a fine physical map of 6P chromosome was constructed, consisting of 31 chromosomal bins with 255 STS markers. Twelve translocation lines containing 6PS13∼14 chromosomal bins were highly resistant to leaf rust. Two lines showed high grain number per spike, and three lines displayed both enhanced grain number per spike and thousand-grain weight. Development of wheat – A. cristatum 6P translocation lines will not only provide novel wheat germplasm for wheat breeding but also be helpful to broaden the genetic basis of common wheat.