Abstract: EST-SSR was used to analyze the genetic polymorphism, relationship, and population structure of Zimeigui, Jinmudan, Mingke 1 (Jinguanyin), Yuemingxiang, and the natural hybrids of Zimeigui, Jinmudan, Jinguanyin, and Yuemingxiang. A total of 125 alleles were amplified using 16 pairs of EST-SSR primers. The average PIC was 0.617, varying from 0.159 to 0.840. The average observed heterozygosity, expected heterozygosity, Nei's genetic diversity, Shannon information diversity index among them were 0.579, 0.639, 0.632, and 1.281, respectively. A strong maternal parental effect seemed apparent as shown by the genetics of the natural hybrids. The population structures of the 4 groups had distinctive genetic characteristics. It was concluded that the software applied could be adequately used for analyzing the genetic structures of tea populations.