Restriction landmark genomic scanning for methylation (RLGS-M) was used to detect, and subsequently clone, genomic regions with alterations in DNA methylation associated with tumorigenesis. Use of a methylation-sensitive enzyme for the landmark cleavage allows analysis of changes in methylation patterns. In this study, we used RLGS-M to analyze SV40 T antigen-induced mouse liver tumors derived from interspecific F1 hybrids between Mus spretus (S) and C57BL/6 (B6). Because 575 S- and B6-specific RLGS loci/spots have been mapped, tumor-related alterations in the RLGS profile could be immediately localized to specific chromosomal regions. We previously found that the loss of contiguous loci/spots could be attributed primarily to DNA loss, whereas loss of solitary loci/spots could be attributed primarily to DNA methylation. In this study, we examined 30 mouse liver tumor samples for loss of the 507 mapped loci/spots. Fourteen solitary loci/spots found to be absent or reduced in more than 75% of tumor samples were cloned and subjected to DNA sequence analyses. Two loci were identified as alpha4 integrin and p16/CDKN2, genes reported to be involved in tumorigenesis. Thus, RLGS-M can detect alterations in the methylation status of known tumor suppressor genes and provide a method for detecting and subsequently cloning novel genomic regions that undergo alterations in methylation during tumorigenesis.