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Background In utero hematopoietic cell transplantation (IUHCT) has been demonstrated to reliably generate chimeric offspring. This technique introduces transplanted cells into a fetus while the immune system is still developing, allowing for engraftment without the need for myeloablation. However, little is known about the effect of engraftment on the gonadal tissue or within the germ line of the resultant chimeras. Materials and methods BALB/cJ mice pups were injected with B6-green fluorescent protein mononuclear bone marrow (BM) cells at gestational ages E13 or E14. Two female and two male chimeras were then crossbred with untreated mice. The gonadal tissue of the chimeras was evaluated with fluorescent stereomicroscopy and green fluorescent protein histologic staining. The progeny of the cross-bred mice was analyzed using flow cytometric evaluation of both the peripheral blood and BM. Results Although transplanted cells engrafted within the gonads, no evidence of chimerism was found in oocytes or spermatogonia of female and male mice treated with IUHCT, respectively. Crossbreeding chimeric mice with untreated mice generated progeny without evidence of chimerism in peripheral blood and BM. Conclusions IUHCT yields chimeric mice that have engrafted cells within the gonads but not within the germ line itself. Correspondingly, progeny from the unaltered germ line has no detectable chimerism. This has clinical implications as the offspring of future patients treated with IUHCT would carry the disease for which their parents were treated with IUHCT.