We analyzed the neighboring-nucleotide composition of 433,192 biallelic substitutions, representing the largest public collection of SNPs across the mouse genome. Large neighboring-nucleotide biases relative to the genome- or chromosome-specific average were observed at the immediate adjacent sites and small biases extended farther from the substitution site. For all substitutions, the biases for A, C, G, and T were 0.21, 2.63, 0.71, and -3.55%, respectively, on the immediate adjacent 5' site and -3.67, 0.75, 2.69, and 0.23%, respectively, on the immediate adjacent 3' side. Further examination of the six categories of substitution revealed that the neighboring-nucleotide patterns for transitions were strongly influenced by the hypermutability of dinucleotide CpG and the neighboring effects on transversions were complex. Probability of a transversion increased with increasing A + T content of the two immediate adjacent sites, which was similarly observed in the human and Arabidopsis genomes. Overall, the bias patterns for the neighboring nucleotides in the mouse and human genomes were essentially the same; however, the extent of the biases was notably less in mice. Our results provide the first comprehensive view of the neighboring-nucleotide effects in the mouse genome and are important for understanding the mutational mechanisms and sequence evolution in the mammalian genomes.