Abstract Ever since its introduction as a genetic tool, the Cre-lox system has been widely used for molecular genetic studies in vivo in the context of health and disease, as it allows time- and cell-specific gene modifications. However, insertion of the Cre-recombinase cassette in the gene of interest can alter transcription, protein expression, or function, either directly, by modifying the landscape of the locus, or indirectly, due to the lack of genetic compensation or by indirect impairment of the non-targeted allele. This is sometimes the case when Cre-lox is used for muscle stem cell studies. Muscle stem cells are required for skeletal muscle growth, regeneration and to delay muscle disease progression, hence providing an attractive model for stem cell research. Since the transcription factor Pax7 is specifically expressed in all muscle stem cells, tamoxifen-inducible Cre cassettes (CreERT2) have been inserted into this locus by different groups to allow targeted gene recombination. Here we compare the two Pax7-CreERT2 mouse lines that are mainly used to evaluate muscle regeneration and development of pathological features upon deletion of specific factors or pathways. We applied diverse commonly used tamoxifen schemes of CreERT2 activation, and we analyzed muscle repair after cardiotoxin-induced injury. We show that consistently the Pax7-CreERT2 allele targeted into the Pax7 coding sequence (knock-in/knock-out allele) produces an inherent defect in regeneration, manifested as delayed post-injury repair and reduction in muscle stem cell numbers. In genetic ablation studies lacking proper controls, this inherent defect could be misinterpreted as being provoked by the deletion of the factor of interest. Instead, using an alternative Pax7-CreERT2 allele that maintains bi-allelic Pax7 expression or including appropriate controls can prevent misinterpretation of experimental data. The findings presented here can guide researchers establish appropriate experimental design for muscle stem cell genetic studies.