Objective The technical process of chicken semen cryopreservation was optimized to solve the poor quality of chicken semen cryopreserved in the Yunnan Plateau regions. Methods The semen of Tea Flower No. 2 and Yunling Red Rooster were used to conduct comparative analyses on the effects of semen diluents with different components, antifreeze agents in different proportions, and methods of freezing on the preservation. The computer-assisted sperm analysis system, Giemsa staining method, and Eosin staining method were used to detect the post-freezing motility of sperm, the integrity rate of sperm acrosome, and the rate of abnormality in chicken semen. Results The effect of formulae 2 liquid, 3 liquid and 7 liquid of basic liquid was better. The post-freezing motility of sperm in the semen froze with GL+10% yolk and DMSO+20% yolk was significantly better than that with DMA+20% yolk and EG+15% yolk, with the rate of 34.1%, 31.6%, 27.4%, and 19.9%, respectively. There was no significant difference in the integrity rate of sperm acrosome. The post-freezing motility of sperm, the integrity rate of sperm acrosome, and the rate of abnormality in semen froze with the programmable instrument and the large-mouthed jar was not significantly different as well. Conclusion It is recommended to use a programmable instrument to prepare frozen semen by adding glucose, lactose, and sodium citrate as the basic liquid solution and EG+15% yolk and DMSO+20% yolk as antifreeze agent. It will provide a theoretical basis for the production and studies of frozen chicken semen in plateau regions.