The primary structure of the Dascyllus trimaculatus mitochondrial (mt) control region was determined, and conserved features were identified based on sequence comparisons to other teleostean fish species. Three termination-associated sequence (TAS) motifs, a central conserved sequence block (CSB), and a pyrimidine tract were identified, indicating that the organization of the mt control region in D. trimaculatus is similar to those reported for other teleosts. However, 2 sets of variable number of tandem repeats (VNTRs), denoted R1 and R2, which simultaneously occur at both the 5' and 3' -ends of the mt control region are first reported herein in teleost fish. Analysis of amplification of the VNTRs indicated that heteroplasmy is common for both sets of VNTRs. VNTRs also respond to sequence length variation of the D. trimaculatus mt control region. A relatively high proportion of VNTR copy number variations and heteroplasmy indicate the potential utility of VNTRs as markers for assessing three-spot dascyllus population-level variability.