Wild-derived mouse strains have been extensively used in biomedical research because of the high level of inter-strain polymorphisms and phenotypic variations. However, they often show poor reproductive performance and are difficult to maintain by conventional in vitro fertilization and embryo transfer. In this study, we examined the technical feasibility of derivation of nuclear transfer embryonic stem cells (ntESCs) from wild-derived mouse strains for their safe genetic preservation. We used leukocytes collected from peripheral blood as nuclear donors without sacrificing them. We successfully established 24 ntESC lines from two wild-derived strains of CAST/Ei and CASP/1Nga (11 and 13 lines, respectively), both belonging toMus musculus castaneus, a subspecies of laboratory mouse. Most (23/24) of these lines had normal karyotype, and all lines examined showed teratoma formation ability (4 lines) and pluripotent marker gene expression (8 lines). Two male lines examined (one from each strain) were proven to be competent to produce chimeric mice following injection into host embryos. By natural mating of these chimeric mice, the CAST/Ei male line was confirmed to have germline transmission ability. Our results demonstrate that inter-subspecific ntESCs derived from peripheral leukocytes could provide an alternative strategy for preserving invaluable genetic resources of wild-derived mouse strains.