The purpose of this paper is breeding base materials with economic value of small fragments on R group for wheat breeding we analyzed 8 high-generation materials of wheat-rye 5R/5A × 6R/6A substitution lines (hybridization 6-30, 6-31, 7-1, 7-9, 7-13, 7-21, 7-22, 7-28) by the morphological and cytological characteristics observation, SSR analysis and GISH detection. The results showed that 8 lines were regular in field growth with normal fertility, big spike, more spikelets, high yield, and resistance to powdery mildew and leaf rust disease as well as other good characters. Meiosis observation of two lines 7-1 and 7-9 showed that most cell chromosome configurations were 2n =21Ⅱand had a good genetic stability. Eight primer pairs located on the rye R chromosome universal primer and 5R,6R chromosome were used for SSR analysis, the results showed that 8 lines contained genetic element of rye 5R or 6R chromosome. Further testing by GISH showed that five strains (6-31,7-1,7-13,7-21, 7-22) had rye hybridization signals and proved to be wheat-rye small fragment translocation lines. Eight materials identified with comprehensive variety of testing means in this study were confirmed to be wheat-rye small fragment translocation lines that have potential utility value in wheat breeding.