Amylomyces rouxii is commonly found as amylolytic fungi in tapai fermentation. However, its diversity is rarely reported despite being often used for food production in Southeast Asia. This research aims to analyze the genetic diversity and the distribution pattern of A. rouxii from Ragi tapai in Java Island, Indonesia. We isolated the fungus from samples obtained from Ragi tapai producing centers in Bandung, Sumedang, Muntilan, Blora, Yogyakarta, and Bondowoso. The obtained isolates were molecularly identified based on the ribosomal regions ITS1/ITS2 and D1/D2, then analyzed for phylogenetic tree reconstruction, genetic distance, genetic variation, and haplotype networking. Six isolates showed specific morphological traits ofA. rouxii. However, phylogenetic tree reconstruction on the ribosomal genes showed that the isolates were grouped into two different clades related to two species. Clade A included BDG, SMD, and MTL isolates related to A. rouxii, whereas clade B included YOG, BLR, and BDS isolates related to Mucor indicus. The genetic distances between clades for ITS1/ITS2 and D1/D2 were 0.6145 and 0.1556, respectively. In conclusion, we confirmed the genetic diversity of molds from Ragi tapai in Java Island and showed that the isolates are not only related to A. rouxii as reported before.