Elevation of scrotal temperature may be injurious to spermatogenesis and leading cause male infertility. Scrotal heat stress reduces the number and motility of spermatozoa, fertilization ability of the surviving sperm and poor fertilization-embryo. This study was designed to investigate the effect of heat stress (at 37 ºC, 40 ºC and 43 ºC) on histopathological features of testicular tissue in scrotal heat exposed male mice. Experimental and control groups were sacrificed after completion of five weeks heat exposure period. The testes were fixed and stained hematoxylin-eosin (H&E) for qualitative and quantitative analysis of histopathological alterations and spermatogenesis according to Johnson scoring system. The results indicated that mice exposed to heat-stress mice exhibited degenerated and disorganized features of spermatogenic epithelium and reduced spermatogenic cells. Heat stress exposure (40 ºC and 43 ºC) shows the significantly reduced Johnson score compared to the control condition (P < 0.05 and P < 0.001, respectively). Meanwhile, scrotal heat exposure at 37 ºC did not reach significantly changes in Johnsen’s testicular histopathological score. The seminiferous tubule structure and spermatogenesis were completely disrupted in mice exposed to 43 °C. Percentage of high Johnsen score point was decreased in mice in heat-stress exposure group, while the ratio of low Johnsen score points was gradually increase. Spermatogenesis in male mice exposed to chronic scrotal heat stress is disrupted at 43 °C. In conclusion, this study attempted to develop an animal model for studying the male reproductive system. Johnsen scores system was standardized to assess murine testicular histopathology in the seminiferous tubule cross-section. Collectively, these results indicated a negative impact on histopathological alterations and spermatogenesis arrest following chronic scrotal heat stress.