The genetic integrity of the European Apis mellifera subspecies in many European regions has been threatened by the human-mediated dispersion of non-native populations and lines, which has intensified over the last few years. Many studies have utilized mitochondrial DNA (mtDNA) analysis from honey bees to gather information on the diffusion of various mtDNA haplotypes and to monitor the genetic integrity of A. mellifera subspecies. Honey contains environmental DNA (eDNA) traces from all organisms directly or indirectly involved in its production, including the DNA of the honey bees that produced it, making it useful for indirectly monitoring the genetic integrity of honey bee populations. In this study we present a snapshot of the diffusion of the main A. mellifera mtDNA lineages in Italy from the last few decades using honey as a source of honey bee mtDNA sequences. DNA was extracted from over 5000 honey samples produced in all regions of the Italian peninsula, Sardinia and Sicily, from 1985 to 2024. PCR products were analyzed using a fragment size-based assay. Results confirmed that the C lineage was the most frequent mtDNA haplotype throughout Italy except in Sicily where the A lineage was highly represented. Other mtDNA haplotypes (A and M lineages) were present in almost all Italian regions. The frequency of the A mtDNA lineage has recently increased in many Italian regions, indicating an increasing level of genetic admixture within the Italian honey bee population. The results provide a time-dynamic distribution map of honey bee mtDNA lineages in Italy that can be valuable for designing and evaluating the potential effectiveness of conservation policies aimed at maintaining the integrity of honey bee genetic resources in Italy. Additionally, we also demonstrated that honey offers a simplified yet extensive population genetic picture, as each honey sample may contain the DNA of many different honey bees from different colonies or even different apiaries.