Abstract: Molecular marker is one of the important ways for precise identification of Camellia oleifera varieties. The genetic diversity of 66 varieties of C. oleifera which were from Hunan and Hubei provinces was investigated using ISSR marker to reveal the affinity of these varieties. The results showed that: 6 ISSR primers selected from 100 primers were employed to conduct PCR amplification on genome DNA of 66 varieties of cultivated C. oleifera. And a total of 121 countable bands were amplified. Among them, the number of polymorphic bands(NPB)were 112, and percentage of polymorphic bands(PPB)was 92.56%. The average value of observed number of alleles, average value of effective number of alleles, Nei’s gene diversity and Shannon’s information index were 1.933 9,1.650 9, 0.369 8 and 0.541 7 respectively. The above parameters indicated that the level of genetic diversity in different varieties of C. oleifera was high. Different varieties of C. oleifera could be identified according to whether the polymorphic bands exist or not, the number of polymorphic bands, and the type of polymorphic bands amplified by different primers.