Haemaphysalis longicornis, the Asian long-horned tick, is an important vector for various infectious diseases, such as severe fever with thrombocytopenia syndrome (SFTS) and Japanese spotted fever. In this species, a triploid parthenogenetic reproductive form occurs along with a diploid bisexual form. Several approaches have been used to distinguish these two groups, including the presence/absence of males in the population, karyotyping, flow cytometry, and most recently, mitochondrial phylogeny. Mitochondrial gene (COI) barcoding has also been casually used, although its validity has not been investigated. In the present study, the validity of COI barcoding, genotyping nuclear markers (SNPs), and morphometrics was evaluated for distinguishing the reproductive forms of H. longicornis in Japan. Ticks were collected using the flagging method at two locations in Hyogo, Japan. DNA was extracted from ticks after photography, which was used for morphometric measurements. The DNA was used for COI barcoding by direct sequencing and genotyping SNPs in the nuclear genome. The resulting COI haplotypes were clustered into two distinct haplogroups, which represented different ploidy levels, corresponding to the different reproductive groups. Genotypes of nuclear SNPs supported that the individuals from each mitochondrial haplogroup belonged to distinct reproductive populations with different ploidy levels. Meanwhile, although significant differences were observed in multiple morphometric characteristics between these reproductive groups, large overlaps were generally evident in the distribution, indicating that morphological identification is not sufficient to distinguish the reproductive groups. This study suggested for the first time that COI barcoding and SNP genotyping are both convenient and reliable methods to distinguish the two reproductive forms of H. longicornis in Japan.