© 2023 Elsevier LtdThis study aims to compare the melissopalynological method with Next-Generation Sequencing (NGS) to determine honey's botanical and geographical origin. For NGS analysis, 40 honey samples in the collected 74 honey samples were selected by melissopalynological methods considering the total number of pollen, honeydew elements, and dominance pollen status. The quality of the isolated DNAs was determined by spectrophotometric and electrophoretic methods. DNA specimens extracted from the 40 honey samples yielded between 2 and 190 ng/μl. We compared NGS data using the rbcL and trnH-psbA DNA barcode marker with melissopalynological analysis. With NGS analysis, 3,020,963 sequence reads and 526,746,371 bases were obtained for 61 amplicons. NGS analysis detected 48% more plant families for all honey samples than the melissopalynological method, showing that NGS is a powerful and robust method for detecting honey provenance. In the conclusion of this study, for the first time, honey samples collected from different geographical regions in Turkey were barcoded using rbcL and trnH-psbA loci and compared using melissopalynology and NGS. Although there are some differences in the plants determined by both techniques, identified dominant floral components were shown high similarity. Results show that 53 families have an abundance of >20% when comparing the two methods. Of these, 29 taxa are found using both methods, giving 55% correspondence between DNA and microscopy. This study is particularly relevant for revealing Turkey's unique and diverse flora, with the potential to be used as a reference atlas for honey bee floral resources.