To provide a reference for the protection and utilization of Liquidambar formosana resources in Hubei Province, L. formosana populations of Yuan'an (YA), Hong'an (HA), Chibi (CB), Zhuxi (ZX), Nanzhang (NZ), Lichuan (LC) and Wuhan (WH) in Hubei Province were adopted as the materials, coupled with the control populations from Huangshan (HS) in Anhui, Fengdu (FD) in Chongqing, Tonggu (TG) in Jiangxi and Bawangling (BWL) in Hainan. The genetic diversity of 335 samples from the 11 populations were analyzed by ISSR (inter-simple sequence repeat). The results were as follows. (1) A total of 334 bands were amplified from the 215 samples of the seven populations in Hubei Province, including 306 polymorphic bands. The genetic variations of intra- and inter-populations were 84.19% and 15.81%, respectively, and the gene flow (N_m) was 2.663 3. Within the population, HA and WH had the most abundant genetic diversity, and ZX had the lowest genetic diversity. The relationship between ZX and LC was the closest, while which between YA and LC was the farthest. The seven populations could be divided into four groups at the genetic similarity coefficient of 0.94. The populations of NZ, LC and ZX were clustered into the first group, YA and WH