This Systematic Literature Review (SLR) consolidates current research on mitochondrial DNA (mtDNA) analysis in horses, focusing on genetic variation, maternal lineage tracing, and haplogroup identification. The article selection process screened 1380 articles, with 76 fulfilling the inclusion criteria. Data extraction covered sampling techniques, studied mtDNA regions, sequencing methods, and haplogroup identification. Following the methodology of the PRISMA guidelines, this review encompasses studies published since 2012, obtained from Scopus, PubMed, Research4Life, Web of Science, and ScienceDirect. The major findings emphasise the use of mtDNA for tracing ancestry, validating maternal lineages, and identifying haplogroups along with their geographic distributions. This review identifies challenges, including the need to update the haplogroup classification system and potential information loss due to sequence trimming. Additionally, it examines promising avenues for future research, such as the implementation of next-generation sequencing and the merging of haplogroup data with performance traits, which could influence conservation initiatives and breeding programs. This review emphasises the necessity for standardised classification systems and further research on underrepresented breeds and regions to improve our understanding of equine genetic diversity.