Systematics of bees has developed rapidly. Several studies have attempted to infer the kinship between each group of bee. One way is the use of phylogenetic analysis using molecular data. This study explains the phylogenetic relationship of stingless bees in the Special Region of Yogyakarta, Indonesia based on the 16S rRNA gene. The research has been carried out in five districts and cities in the region from June to September 2019. In the study, the stages implemented include; place determination of stingless bees sampling, sampling, and molecular identification (DNA extraction, DNA amplification, and sequencing), followed by the data analysis using NCBI database and MEGA X software. The result of this study indicated that among seven morphospecies from Yogyakarta, there are six species of stingless bees that have a closest genetic relationship with the same species data from Genbank, namely Tetragonula laeviceps, T. iridipennis, T. sapiens, T. sarawakensis, Lepidotrigona terminata, and Heterotrigona itama. Since the genetic distances of T. laeviceps, T. iridipennis, T. sapiens, are wider than 3,5%, the data indicate that there is a possibility that the three morphospecies are actually belong to a different species with a similar morphology. Meanwhile, for the morphospecies T. biroi, the closest hit is on T. pagdeni 16S rRNA DNA because the T. biroi 16S rRNA DNA data is not available on the database. This study is expected to contribute to the preservation and utilization of one of Indonesia's important biodiversity resources.