Camellia oleifera is an important woody plant producing healthy edible oils. People need a large number of molecular markers, especially microsatellite, in breeding of C. oleifera. In this study, we sequenced the root transcriptomes of C. oleifera, and then designed a novel set of microsatellite markers based on the root-expressed genes. We assembled a total of 57,121 unigenes with a length of 42.63 Mb, which harboured 15,902 microsatellites. Among these microsatellites, di-nucleotide repeat motifs were the most abundant group (56.45%), then followed by tri- (25.20%), mono- (12.12%), hexa- (3.21%), penta- (2.18%) and quad-nucleotide ones (0.84%). In total, 6738 primer pairs were designed successfully to amplify the microsatellite loci. To test these microsatellite markers, 48 primer pairs were randomly selected and synthesized and validated in C. oleifera and its eight relatives. Up to 75% of the primer pairs amplified in C. oleifera and its relatives, and 62.5% displayed polymorphism. The transferability and diverse alleles across its eight relatives were detected for each polymorphic primer pair. The novel set of microsatellites derived from the root transcriptomes here provided a useful resource for future molecular genetics improvement of C. oleifera and its relatives.