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The C57BL/6 mouse is the most well-known inbred mouse strain, and has been widely used as a genetic background for congenic and mutant mice. A number of C57BL/6 substrains have been derived from the C57BL/6 founder line and are reported to differ in several phenotypes. There are several major sources of C57BL/6 substrains for the biomedical research community. The importance of their genetic and phenotypic differences among substrains, however, has not yet been well recognized by biomedical researchers. Here, we report the result of screening of the functional deletion of the nicotinamide nucleotide transhydrogenase (Nnt) gene and 1,446 SNPs genotyping among seven C57BL/6 substrains from different sources, such as C57BL/6J, C57BL/6JJcl, C57BL/6JJmsSlc, C57BL/6NJcl, C57BL/6NCrlCrlj, C57BL/6NTac, and C57BL/6CrSlc. The deletion of exon 7-11 in the Nnt gene that was previously reported in C57BL/6J was also observed in other C57BL/6J substrains, indicating that this functional deletion probably occurred at an early stage in the establishment of C57BL/6J substrains. The genotyping of SNP loci clearly demonstrate genetic differences between C57BL/6J and C57BL/6N substrains at 11 loci. Besides, we found another SNP differing between C57BL/6J and other C57BL/6J substrains available from commercial breeders. No genetic difference was detected among C57BL/6N substrains. The C57BL/6CrSlc mouse, originally derived from the National Cancer Institute of the NIH was found to be the same as the C57BL/6N substrains by the SNP pattern. These data will be useful for accurate genetic monitoring of genetically engineered mice with the C57BL/6 background.