Oryza rufipogon Griff., or “Rufi” in the Philippines, was previously known to be found only in Lake Apo, Bukidnon. However, a new population was identified in Lake Napalit in the same province. A better understanding of the genetic diversity of both Rufi populations using molecular methods may be beneficial to further ascertain its usefulness in rice breeding and in the development of effective conservation strategies. Population genetic analysis was conducted to estimate the degree of genetic diversity and population structure of the two Philippine Rufi populations using 98 genome-wide simple sequence repeat (SSR) markers. Four Oryza sativa indica and three O. sativa japonica cultivars were added for comparison. Results indicate that both Rufi populations exhibit low genetic diversity but with significant population structure and differentiation. Low genetic diversity suggests that both populations might be in a genetic bottleneck, perhaps due to observed unsustainable farming practices near their habitat and lack of awareness among locals of their importance. Also, geographical isolation that prevented gene flow between the two populations, as well as the unique climatic conditions between the two lakes might have contributed to the significant population structuring and differentiation. Thus, in situ and ex situ conservation should be observed for both Rufi populations in the Philippines.