The largemouth bass Micropterus salmoides consists of two subspecies of the northern subspecies M. s. salmoides and the Florida subspecies M. s. floridanus. It has been widely regarded that largemouth bass, introduced and widespread in Japan, is the northern subspecies. In 1988, the Florida subspecies was also introduced into the Ikehara Reservoir (Nara Prefecture), where northern subspecies were already present. To assess the influence of this introduction, the genetic structure of the largemouth bass population in Ikehara Reservoir and thirteen other locations throughout Japan were examined and compared by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for mitochondrial DNA (mtDNA). Analysis of the ND1 region of mtDNA using sixteen restriction enzymes revealed 10 haplotypes (1-10) that form two distinct clusters in the UPGMA tree. One cluster consisted of two haplotypes (1 and 2) commonly detected in most populations throughout Japan, and the other cluster consisted of eight haplotypes (3-10) detected only in Ikehara and its connected Reservoirs. The reported rapid assay to identify mtDNA from two subspecies using D-loop region clearly revealed that eight haplotypes (3-10) were from the Florida subspecies.