Abstract: 【Objective】 The purpose of the study was to clarify the population genetic structure and genetic diversity of Apis cerana in Guizhou，to understand its genetic status，and to provide a reference basis for the evaluation，conservation and utilization of Apis cerana genetic resources in Guizhou.【Method】Genetic analysis was conducted on 1282 populations of A. cerana from 19 sampling sites in Guizhou based on mitochondrial tRNA^leu-COII and COI fragments. Genetic diversity indexes such as haplotype diversity（H_d），the average nucleotide difference（K）and nucleotide diversity（π）were calculated. Tajima’s D and Fu’s Fs neutral tests were carried out to evaluate population expansion. Genetic differentiation was assessed by calculating the genetic differentiation coefficient（F_st），constructing phylogenetic evolutionary trees and molecular variance analysis，and environmental factor correlation analysis was carried out to clarify the role of factors in genetic differentiation.【Result】The H_d of A. cerana in Guizhou on the mitochondrial tRNA^leu-COII fragment ranged from 0.5421 to 0.9167，π ranged from 0.00174 to 0.00527，and K ranged from 0.611 to 1.854，with colony expansions occurring in seven sampling sites，including Taijiang，Leishan，Majiang，Luodian，Ziyun，Qinglong，and Weining. On the mitochondrial COI fragment，H_d ranged from 0.5710 to 0.9109，π ranged from 0.00109 to 0.00261，and K ranged from 0.874 to 2.084，with population expansion occurring at 11 sampling sites，including Songtao，Jiangkou，Yinjiang， Taijiang， Shiqian， Leishan， Luodian， Ziyun， Qianxi， Qinglong， and Weining. There existed moderate genetic differentiation（on tRNA^leu-COII fragment，F_st=0.04±0.02，on COI fragment F_st=0.07±0.04）between A. cerana populations in the eastern and western of Guizhou，and moderate genetic differentiation（on tRNA^leu-COII fragment，F_st=0.04±0.02，on COI fragment，F_st=0.07±0.03）within A. cerana populations in the western part of Guizhou. The results of correlation analysis showed that genetic differentiation of A. cerana colonies in western Guizhou was weakly correlated with altitude，but significantly positively correlated with environmental factors such as temperature and humidity.【Conclusion】The genetic diversity of A. cerana in Guizhou is at a high level in mitochondrial tRNA^leu-COII and COI fragments，which is mainly attributed to the huge population size and primitive rearing methods. Guizhou A. cerana as a whole show moderate genetic differentiation，and the degree of genetic differentiation within the western A. cerana populations is higher than that of the eastern A. cerana，which is mainly closely related to the environmental factors，such as temperature and humidity，and arises from their evolution to adapt to the environment they live in rather than due to altitudinal factors.