Abstract <p>Native honeybee populations are declining worldwide due to habitat loss caused by human activity, hybridization, the spread of diseases, and pesticide use. Natural areas, particularly protected areas, are crucial for the conservation of wild honeybee populations in their natural range. This study presents the results of monitoring the gene pool of the dark forest honeybee, <i>Apis mellifera mellifera</i>, in protected and unprotected areas of the Republic of Bashkortostan (RB). Using genetic markers of mitochondrial and nuclear DNA, the level of introgression of the gene pool of the evolutionary lineage C was determined in <i>A. m. mellifera</i> samples from protected (Shulgan-Tash State Nature Reserve and Altyn Solok State Nature Reserve) and unprotected (apiaries from the Burzyansky and Yanaulsky districts) populations. As a result, we found that the bee sample in the protected area of the Burzyansky district had a lower level of hybridization compared to the sample collected outside protected areas. The lowest level of hybridization with the C evolutionary lineage was recorded in the unprotected sample from the Yanaulsky district of the RB—3.6 and 3.3%, according to analysis of nuclear loci and a mitochondrial marker, respectively. We hypothesize that the natural preservation of this population is facilitated by the presence of an isolation zone in Perm Krai, Udmurtia, and neighboring districts of the RB, where the dark forest bee predominantly inhabits. While honeybee populations bordering the Burzyansky district are of hybrid origin, the majority of colonies are descended from the C evolutionary lineage, based on analysis of the mitochondrial DNA locus.</p>