AbstractHybrid male sterility (HMS) is a reproductive isolation mechanism limiting the formation of fertile offspring through interspecific fertilization. Cattleyak is the interspecific hybrid presenting significant heterosis in several economic traits, but HMS restricted its wide reproduction in cettleyak breeding. In this study, we detected the specifically expressed genes of a variety of cells (undifferentiated spermatogonia, primary spermatocytes, secondary spermatocytes, haploid spermatids, sperm, Sertoli cells, Leydig cells, and macrophages) in the testis of yak and cattleyak, and found that the spermatogenesis of cattleyak might be blocked at meiosis I, and the expression of niche factors (NR5A1, GATA4, STAR, CYP11A1, CD68, TNF, and CX3CR1) in undifferentiated spermatogonia niche was abnormal. Then we isolated the undifferentiated spermatogonia and Sertoli cells from yak and cattleyak by enzyme digestion, and detected the specific genes in the two bovid testicular cells as well as the proliferation capacity of the undifferentiated spermatogonia. These results indicated that weak proliferation ability and scarce number of undifferentiated spermatogonia and abnormal gene expressions in Sertoli cells may contribute to male sterility of cattleyak.