Pepper (Capsicum annuum L.) has been widely used for multiple purposes, including medicine, culinary, and flavoring ingredients. This plant is extensively grown in Vietnam, encompassing diverse climatic conditions and ecosystems. However, there is a lack of understanding regarding the distinctions between most of the local species. Therefore, this study aimed to assess the genetic diversity and relationships of 25 Vietnamese native pepper species collected from two provinces, Quang Nam and Gia Lai, using molecular markers. Specifically, genome sequencing was conducted on the sequences of the internal transcribed spacer (ITS) regions ITS1-5.8S-ITS2 and rbcL regions using two pairs of specific primers: ITS1/ITS8 and rbcL-F/rbcL-R. The results revealed that all the native pepper species are diversified within the genus Capsicum, exhibiting species adaptation and locality-dependent characteristics. The similarity coefficient ranged from 81.27% to 98.99% for the ITS regions and from 97.91% to 100% for the rbcL region. These findings provide a foundation for identifying valuable genetic resources that can contribute to the development, breeding, and conservation of different native types of pepper in this country.