A stable, highly fertile wheat Secale africanum substitution line LF24, derived from the F7 generation of a cross between Mianyang11 (MY11) and Triticum durum, S. africanum amphiploid (YF) was identified through molecular cytogenetic analysis. Application of C-banding, in situ hybridization and molecular markers analysis showed that LF24 was a wheat S. africanum 2R(a)(2D) substitution line. When inoculated with stripe rust isolates, T. durum and MY11 were highly susceptible, while S. africanum, YF and LF24 were immune. It is confirmed through molecular cytogenetic analysis that the stripe rust resistance of LF24 was derived from S. africanum chromosome 2R(a). We compared the banding patterns and disease resistance of reported chromosomes 2R from different S. cereale introduced into wheat background, and found that there was new stripe rust resistance gene(s) on S. africanum 2R(a). LF24 is a new substitution line which can be used as stripe rust resistant source in wheat improvement.