The Cre‐LoxP system gene knockout (KO) technology provides cell‐ and time‐specificity of gene ablation to investigate cell‐autonomous gene function in vivo, and is paramount for understanding the function of genes involved in bone development, remodeling, and repair. This approach permits gene ablation in a cell‐ or tissue‐specific, differentiation stage‐specific, and inducible manner, thanks to the use of well‐chosen promoters that drive expression of the Cre recombinase in selected cells/tissues. The generation of these powerful tools has led to the expansion of Cre mouse lines available to the research community, which are often shared within and between laboratories. Although convenient and commonly used, genotyping these Cre lines with a generic set of primers that amplifies the Cre transgene does not distinguish between various Cre‐deleter lines. This practice poses the significant risk of mistakenly swapping Cre lineages, as laboratories often host and handle several lines at a time and utilize multiple lines per project. In line with the NIH‐led effort to promote authentication of biological reagents and increase scientific rigor, we report here strategies for designing appropriate sets of primers able to discriminate some of most widely used Cre‐deleter mouse lines in the field of bone biology, and the validation of 24 of them. © 2018 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.