F1 hybrid progenies between related subspecies often show hybrid sterility (HS) or inviability. HS is caused by failure of meiotic chromosome synapsis and sex body formation in house mouse. Previous studies identified two HS critical genomic regions named Hstx2 on Chr X and Hst1 on Chr 17 by murine forward genetic approaches. HS gene on Hst1 was reported to be Prdm9. Intersubspecific polymorphisms of Prdm9 induce HS in hybrids, and Prdm9 null mutation leads to sterility in the inbred strain. However, HS gene on Hstx2 remains unknown. Here, using knock-out studies, we showed that HS candidate genes on Hstx2 are not individually essential for spermatogenesis in B6 strain. We examined 12 genes on Hstx2: Ctag2, 4930447F04Rik, Mir743, Mir465d, Mir465c-2, Mir465b-1, Mir465c-1, Mir465, Gm1140, Gm14692, 4933436I01Rik, and Gm6812. These genes were expressed in adult testes, and showed intersubspecific polymorphisms on expressed regions. This first reverse genetic approach to identify HS gene on Hstx2 suggested that the loss of function of any one HS candidate gene does not cause complete sterility, unlike Prdm9. Thus, the mechanism(s) of HS by the HS gene on Hstx2 might be different from that of Prdm9.