Societal Impact Statement: Clarifying species status for closely related taxa with similar and overlapping morphology is important for planning conservation efforts. Conservation of Rafflesia goes beyond merely saving species from extinction. It has far-reaching societal impacts that touch on scientific discovery, cultural heritage, economic development, education, and more. Rafflesia interests many nature lovers, and so protected areas that preserve Rafflesia can become tourism hotspots, contributing to local economies and livelihoods. By conserving biodiversity, we acknowledge and value the interdependence of all living organisms, promoting a holistic understanding of nature. Biodiversity conservation is an investment in the health and well-being of both ecosystems and human societies. Summary: Species ambiguity could hinder conservation activities. Rafflesia is an endangered, holoparasitic, and endophytic plant genus that grows exclusively on Tetrastigma (Vitaceae) host plants. Overlapping morphological characters make their species delimitation difficult. Therefore, the number of Rafflesia species has been a major debate until now. On the island of Java, Indonesia, two or three species have been variously recognized. Here, we aim to illuminate the species status using single nucleotide polymorphisms (SNPs) marker obtained via a multiplexed ISSR genotyping by sequencing (MIG-seq) approach. Clustering analysis with STRUCTURE indicates three different groups. Furthermore, the delimitation of the Javanese Rafflesia into three species is the favored model based on SNAPPER analysis. Phylogenetic analysis using Randomized Axelerated Maximum Likelihood (RAxML) also supports the distinction of the three groups. Although a SplitsTree4 analysis confirms the abovementioned results, it also highlights that Rafflesia patma shows broader gene flow compared to the other two species R. rochusenii and R. zollingeriana. We conclude that there are likely three species of Rafflesia on Java. Our study highlights the ability of using SNPs markers, in this case derived from a MIG-seq approach, in approaching species uncertainty. The importance of clarifying the Rafflesia species status on Java Island for conservation planning is also discussed.