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Pinus kwangtungensis (Pa Co pine) is one of three five-needle pine species in Vietnam, found on the slopes of limestone mountains at altitudes between 1200 and 1500 m. Global warming and long-term deforestation threaten the existence of the species in nature. The genetic diversity of plant populations provides a background for future conservation and improvement programmes. However, the genetic diversity of Pa Co pine is unknown. This study aimed to use inter-simple sequence repeat (ISSR) and random amplified polymorphic DNA (RAPD) genetic markers to evaluate the genetic-diversity parameters of P. kwangtungensis to understand the genetic effects of small and fragmented populations, as well as provide the genetic background for its conservation. Total genomic DNA was extracted from fresh needles of 40 trees in four different areas and amplified with 15 RAPD and 16 ISSR markers. Results indicated that the genetic diversity index (h) of P. kwangtungensis was 0.2530 with RAPD and 0.223 with ISSR. High genetic variation was found within populations (72% with RAPD and 87% with ISSR). Principal coordinates analysis based on RAPD analysis revealed that the presence of three groups was in accordance, whereas no clear cluster was formed according to ISSR analysis. The results from this study enhance the understanding of the genetic effects of small and fragmented populations of native species that are rare, vulnerable, and require conservation.