Honey is a natural product widely consumed globally for its associated health benefits. This food product is also linked with environmental issues due to the importance of honeybees in biodiversity preservation. Consequently, various methods have been proposed and developed to assess the quality and origin of honey and to study environmental events. DNA-based approaches have gained particular attention for their utility in discerning the geographical, botanical, and entomological origins of honey. Additionally, different DNA markers have been explored to study diverse sources of honey DNA, aiming to assess the quality of the honeycomb's surrounding environment and monitor the presence of invasive organisms [1]. The aim of this work was to evaluate different target genes for the amplification of plant species from genera Lavandula spp., Quercus spp., and Castanea spp., as well as the ectoparasite Varroa destructor, in Portuguese honey samples. Honey samples from distinct regions of Portugal were acquired from producers and supermarkets. After DNA extraction [2], specific PCR amplifications were performed with primers targeting different genes, namely, the plastidial matK gene of Lavandula stoechas L. [2], the rbcL gene of Quercus pyrenaica (designed in this work), and the gene COX1 no mtDNA of Varroa destructor [3]. The results indicate the potential of primers targeting matk and rbcL genes in identifying three of the major plant genera found in Portuguese honey: Quercus spp., Castanea spp., and Lavandula spp. Additionally, the application of primers selected for the amplification of Varroa destructor demonstrated potential for detecting the presence of this ectoparasite in honey samples.