Mouse embryogenesis relies on the presence of both the maternal and the paternal genome for development to term. It has been proposed that specific modifications are imprinted onto the chromosomes during gametogenesis; these modifications are stably propagated, and their expression results in distinct and complementary contributions of the two parental genomes to the development of the embryo and the extraembryonic membranes. Genetic data further suggest that a substantial proportion of the genome could be subject to chromosomal imprinting, the molecular nature of which is unknown. We used random DNA insertions in transgenic mice to probe the genome for modified regions. The DNA methylation patterns of transgenic alleles were compared after transmission from mother or father in seven mouse strains carrying autosomal insertions of the same transgenic marker. One of these loci showed a clear difference in DNA methylation specific for its parental origin, with the paternally inherited copy being relatively undermethylated. This difference was observed in embryos on day 10 of gestation, but not in their extraembryonic membranes. Moreover, the methylation pattern was faithfully reversed upon each germline transmission to the opposite sex. Our findings provide evidence for heritable molecular differences between maternally and paternally derived alleles on mouse chromosomes.