Targeted mutagenesis in mice, a powerful tool for the analysis of gene function and human disease, makes extensive use of 129 mouse substrains. Although all are named 129, we document that outcrossing of these substrains, both deliberate and accidental, has lead to extensive genetic variability among substrains and embryonic stem cells derived from them. This clearer understanding of 129 substrain variability allows consideration of its negative impact on targeting technology, including: homologous recombination frequencies, preparation of inbred animals, and availability of appropriate controls. Based on these considerations we suggest a number of recommendations for future experimental design.