Spontaneous mutations originate evolution. Kimura’s neutral theory elucidated the fixation rate of neutral mutations to be equal to the mutation rate of 1X10-8/yr. On the other hand, mutation rate, encompassing neutral mutation rate, varies depending on gender, genetic background, environmental factors and so forth. The estimation of mutation rate is thus critical for the studies on evolution as well as for the assessments of environmental risk factors. The advancement of high-throughput sequencing technologies have made it possible to comprehensively detect and analyze very rare mutations even in mammalian genomes. We here designed a mating scheme to accumulate germline spontaneous mutations, which is universally applicable to any sexually reproductive species. Starting from twelve sets of G1 mating pairs of C57BL/6JJcl inbred mice, we obtained eight G5 females from eight independent pedigrees in which de novo germline spontaneous mutations were accumulated by complete outbreeding. The genomic DNA samples of the eight G5 female mice were subjected to whole genome sequencing (WGS). The single nucleotide variation (SNV)-call informatics pipeline subjected to the WGS dataset primarily gave rise to a total of 11,984 SNV candidates. These SNV calls consist of not only de novo spontaneous germline mutations that arose somewhere between G1 gametogenesis and G5 fertilization but also SNVs/SNPs predisposed in the twelve sets of the original G1 genomes. For instance, 4,601 primary SNV calls were found to be homozygous in all the eight G5 females; thus, they are the fixed SNPs between C57BL/6J and C57BL/6JJcl strains. The two inbred strains were branched in 1989 and the current mouse genome reference sequence (GRCm38.4) of C57BL/6J is 2.8 Gbps; thus, ~1.6 base substitutions and fixation per Mb on average have occurred in a total of 50 yrs (= 25 yrs X 2) or 3.3X10-8/bp/yr. One thousand nine hundreds and fifty six SNV calls were found in two or more of the eight G5 females but not completely fixed yet; thus, they are recently-arisen polymorphic SNVs predisposed to the breeder’s colony. The remaining 5,427 SNV candidates were identified in only one G5 female as heterozygotes; thus, they are good candidates for the de novo germline spontaneous mutations accumulated in the four generations between G1 and G5. We are currently focusing on the validation and identification of the origin of the 5,427 SNV candidates in the eight outbred pedigrees.