To search for hyper-variable genetic markers that can distinguish regional populations of head lice, we screened the inter simple sequence repeats (ISSR) based on the genome database of body louse, which is closely related conspecific species. An ISSR mining software, SciRoKo 3.4, was employed to excavate ISSR markers from the genome database under the MISA mode (≥ 60 bp repeats). Entire body louse genome (ca.100 Mb) was loaded to SciRoKo for ISSRs mining. A total of 5,336 ISSRs were obtained, and primers specific to individual ISSRs were designed by the Primer 3 and DesignPrimer 1.0 softwares. In order to prove the compatibility of body louse ISSRs to head lice, 31 PCR primers were randomly chosen out of a total of 613 pairs, and their appropriateness was tested by comparing the amplified PCR band patterns between body and head lice. Eleven primer pairs that resulted in poor or little amplification were excluded, and 20 primer pairs were further tested for three head louse populations (California, Panama and Chung-ju, Korea). Finally, nine primer pairs ensuring robust amplification of highly variable band patterns were selected to use for population genetic study of head lice.