Intergeneric hybridization is an important strategy to introgress alien genes into common wheat for its improvement. But presence of crossability barrier mechanism regulated by Kr1 gene played a major destructive role for hybridization than other reported genes. In order to know the underlying molecular mechanism and to dissect out this barrier, cDNA-AFLP (Amplified Fragment Length Polymorphism) transcriptional analysis was carried out using chromosome 5B RIL (Recombinant Inbred Line) population. Totally 14 differentially expressed fragments for Kr1 gene were identified, cloned and sequenced. Further the expression was confirmed by northern blotting analysis. Sequence analysis of the resulted clones revealed several classes of putative genes, including stress responsive, signal transduction and flowering related genes. The probable relation between other genes, Vrn1 and Ph1, located along with Kr1 gene was discussed.