The present study was designed to evaluate the longevity of canine cauda epididymal sperm in vitro at 40C. The testes were collected from 30 mixed breed dogs of average weight of 20-25Kg,presented to the clinics for sterilization. The testes were stored at 40C in normal saline in a refrigerator.The sperms were collected from the epididymis at different intervals of time (0, 4, 8, 12 and 24 hr) by taking 6 testes in each interval of time. The sperm quality was accessed at different intervals of time.The data analysed was analysed by ANOVA and the significant level was 5% (P= 0.05). The sperm concentration at different interval of time and storage at 40C did not show significant difference. On the other hand the sperm motility revealed significant difference between all the time intervals of sperm collection except at 8 and 12 hr of collection. There was significant difference of live sperm (%) at different intervals of sperm collection except at 8-12 hrs of sperm collection. The percentage of normal sperms was significantly different in between 0 to 24 hr interval of time but there was no significant difference either at 4 and 8hr or 12 and 24 hr of sperm collection. The acrosomal integrity was found statistically significant between 0-24 hrs of sperm collection and storage at 40C. There was significant difference in percentage of hypo- osmotic swelling between 0 hr (66.67 ± 2.11%), 4 hr (40.50 ± 6.21%), 8 hr (26.27 ± 2.25), 12 hr (11.50 ± 1.38%) and 24 hr (6.67 ± 1.05%). However, there was no difference between 8 and 12 hr of sperm collection from testes and storage at 40 C. The study concluded that the cauda epididdymal spermatozoa can be effectively stored and utilised for the purpose of assisted reproductive technology during emergency. These findings can be applied to the endangered wild canines and felines.