Abstract: The band differences of esterase isoenzymes of 25 Kudingcha germplasm materials in Aquifoliaceae, which involved 5 species, i.e. Ilex kudingcha C. J. Tseng, I. latifalia Thunb., I. cornuta Lindl., I. pentagona S. K. Chen, Y. X. Feng et C. F. Liang and I. houshanensis Y. H. He, were analysed via polyacrylamid gel electrophoresis. The similarity coefficients of the test materials, were calculated, and cluster dendrogoam for 25 germplasm materials was drawn by using UPGMA. The experimental results showed that there were 18 bands of esterase isoenzyme observed in all test materials. Of all bands, some of them were the common bands for the all test materials, some for all materials of the same species, and the others showed the genetic differences between different materials. Based on the band differences of the esterase isoenzymes, the 25 test germplasm materials from 5 species in Aquifoliaceae could be obviously distinguished on the species level. The bands of esterase isoenzyme of the test germplasm materials from the same species in Aquifoliaceae showed both abundant polymorphism and relatively obvious similarity. The similarity coefficient of the 25 test germplasm materials, based on the bands of esterase isoenzymes, ranged from 0.1333 to 1.0000. In the cluster analysis dendrogram, the 5 Kudingcha species in Aquifoliaceae could be clearly distinguished, which showed the relationships among the 5 species. The different germplasm materials from the same species were clustered according to their origins basically, which also showed the obvious relation relative to the morphological characteristics. The analysis results of the esterase isoenzyme could be considered as the significant reference bases for the classification of the germplasm materials of Kudingcha species in Aquifoliaceae on the species level, and could be used for determining the origination region, genetic difference, relationships of the germplasm materials of Kudingcha species in Aquifoliaceae.