A few national or regional legislative acts or initiatives have been introduced in Europe to preserve the genetic integrity of native honey bee subspecies. In Italy, for example, the region Emilia-Romagna (North of Italy) has been one of the first regional authorities that issued a regional law, specifically addressed for this purpose, focused on the conservation of the genetic integrity of Apis mellifera ligustica, banning the breeding and introduction in the region of other subspecies. The relevance of this regional law is derived by the fact that this region hosts one of the highest concentrations in the world of the queen breeding activities for A. m. ligustica. In this study, we monitored the distribution of mitochondrial DNA (mtDNA) lineages in a total of 1143 honey bees, each collected from different colonies, taken from apiaries spread all over the region Emilia-Romagna and over three years (2020-2022), after the entry of the mentioned regional act. The informative mtDNA region spanning the tRNALeu-COII genes was sequenced for all these samples and a total of 14 different mtDNA haplotypes (mitotypes) were identified. The most frequent haplotype (86.5%) was C1, which is characteristic of A. m. ligustica. The second mitotype considered to characterize this subspecies (M7) was identified in very few samples (<1%). This study indicated that the regional act seems to be effective in strengthening the conservation of A. m. ligustica genetic pool.