Mitochondrial DNA (mtDNA) is a useful tool in forensic investigation as it provides information about the matrilineal ancestry of individuals. In addition, mtDNA can be analyzed when the analysis of other nuclear markers is underperforming. Recently, we developed a minisequencing panel for the simultaneous analysis of 52 mtDNA SNPs to classify maternal lineages into the main haplogroups and their phylogeographic origin. In order to make this panel suitable for forensic genetics laboratories, a validation study has been performed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, including species specificity, reproducibility, sensitivity, and stability tests. The results demonstrate that the panel of 52 mtDNA SNPs is highly sensitive, since it enables to obtain complete genetic profiles of samples containing minimal amounts of DNA (1 pg). Furthermore, it provides sufficient genetic information to detect the matrilineal biogeographical origin of highly degraded samples, i.e., ancient dating skeletal remains, and samples with the presence of inhibitors, such as hematin and humic acid. In addition, this panel can detect mixtures in samples whose mtDNA haplogroups of contributors are different. Overall, the results of this study demonstrate the suitability of this minisequencing panel of 52 mtDNA SNPs to be used in forensic cases, with samples of low amount or degraded DNA.