Little Tern Sternula albifrons sinensis was been categorized as Endangered Class II by the 2020 Ministry of the Environment's Red List in Japan. For an effective conservation program for the taxon, the unit of conservation needs to be understood. In this study, we investigated genetic and morphological diversity as a clue to clarify the units to be protected. Blood samples were collected from 47 individuals in four different breeding sites in Japan (Tokyo, Chiba, Fukuoka, Okinawa). The nucleotide sequences of the control region of mitochondrial DNA were used to analyze their genetic diversity and structure. Basic morphological features were also compared. Nucleic acid base substitution, deletion, or insertion were identified at 30 sites of the 1029-bp sequence alignment of the control region, leading to identification of 30 different haplotypes. Genetic diversity was high in the four breeding populations, and haplotype frequencies showed no indication that specific genotypes were present at extreme frequencies. This suggests that the population of Little Tern nesting in Japan has been maintained without recent bottleneck events. The haplotype network formed two major haplotype groups from the 30 haplotypes. Haplotypes of one group were abundant in the Tokyo, Chiba, and Fukuoka populations. In contrast, those of the other group were frequent in the Okinawa population. Pairwise haplotype analysis between populations also revealed that the genetic variations at Okinawa differ from those of the remaining three populations. Furthermore, the Okinawa population showed different morphological characteristics compared to the Tokyo/Chiba populations. These results collectively suggest that the population of Little Terns in Japan is divided into at least two different populations.