Abstract: Cytochrome b gene mitochondrial DNA was used to study the Palo fish from Bukit Rangkak tributary, Harau Valley, West Sumatra. The study aimed to determine the taxonomy of Palo fish, which morphological suspected as the Betta group. Phylogenetic analysis was used to solve the relationship of Palo fish with other species of the Betta. The alignment of the DNA sequences was carried out with Clustal X version 2 and analysis of phylogenetic tree using MEGA 6 software program. Based on the analysis of the cytochrome b gene sequence (1047 bp), it is known that the genetic differences of Palo fish from two tributaries of Bukit Rangkak river is 0.0% and with other Betta fish ranges from 13.0-35.5%. The phylogenetic tree has shown that Palo fish has a close genetic relationship with the Betta picta (13.0%). The result showed that Palo fish is at the different species in the genus of Betta and proposed as a new species.