Ixodid ticks (Acari:Ixodidae) are essential vectors of tick-borne diseases in Japan. In this study, we characterized the population genetic structure and inferred genetic divergence in two widespread and abundant ixodid species, Ixodes ovatus and Haemaphysalis flava. Our hypothesis was that genetic divergence would be high in I. ovatus because of the low mobility of their small rodent hosts of immature I. ovatus would limit their gene flow compared to more mobile avian hosts of immature H. flava. We collected 320 adult I. ovatus from 29 locations and 223 adult H. flava from 17 locations across Niigata Prefecture, Japan, and investigated their genetic structure using DNA sequences from fragments of two mitochondrial gene regions, cox1 and the 16S rRNA gene. For I. ovatus, pairwise FST and analysis of molecular variance (AMOVA) analyses of cox1 and 16S sequences indicated significant genetic variation among populations, whereas both markers showed non-significant genetic variation among locations for H. flava. A cox1 gene tree and haplotype network revealed three genetic groups of I. ovatus. One of these groups consisted of haplotypes distributed at lower altitudes (251–471 m.a.s.l.). The cox1 sequences of I. ovatus from Japan clustered separately from I. ovatus sequences reported from China, suggesting the potential for cryptic species in Japan. Our results support our hypothesis and suggest that the host preference of ticks at the immature stage may influence the genetic structure of the ticks. This information may be important for understanding the tick-host interactions in the field to better understand the tick-borne disease transmission and in designing an effective tick control program.