To study the possible impact of alien introgression on a recipient plant genome, we examined >6000 unbiased genomic loci of three stable rice recombinant inbred lines (RILs) derived from intergeneric hybridization between rice (cv. Matsumae) and a wild relative (Zizania latifolia Griseb.) followed by successive selfing. Results from amplified fragment length polymorphism (AFLP) analysis showed that, whereas the introgressed Zizania DNA comprised <0.1% of the genome content in the RILs, extensive and genome-wide de novo variations occurred in up to 30% of the analyzed loci for all three lines studied. The AFLP-detected changes were validated by DNA gel-blot hybridization and/or sequence analysis of genomic loci corresponding to a subset of the differentiating AFLP fragments. A BLAST analysis revealed that the genomic variations occurred in diverse sequences, including protein-coding genes, transposable elements, and sequences of unknown functions. Pairwise sequence comparison of selected loci between a RIL and its rice parent showed that the variations represented either base substitutions or small insertion/deletions. Genome variations were detected in all 12 rice chromosomes, although their distribution was uneven both among and within chromosomes. Taken together, our results imply that even cryptic alien introgression can be highly mutagenic to a recipient plant genome.