Expression regulation of the Dlk1-Dio3 imprinted domain by the intergenic differentially methylated region (IG-DMR) is essential for normal embryonic development in mammals. In this study, we investigated conserved IG-DMR genomic sequences in eutherians to elucidate their role in genomic imprinting of the Dlk1-Dio3 domain. Using a comparative genomics approach, we identified three highly conserved sequences in IG-DMR. To elucidate the functions of these sequences in vivo, we generated mutant mice lacking each of the identified highly conserved sequences using the CRISPR/Cas9 system. Although mutant mice did not exhibit the gross phenotype, deletions of the conserved sequences altered the expression levels of paternally expressed imprinted genes in the mutant embryos without skewing imprinting status. These results suggest that the conserved sequences in IG-DMR are involved in the expression regulation of some of the imprinted genes in the Dlk1-Dio3 domain.