Magnolia patungensis (Magnoliaceae) is an endangered tree species endemic to China. To facilitate analysis of its genetic variability, we developed novel polymorphic expressed sequence tag-simple sequence repeat (EST-SSR) markers using transcriptome data. In total, 37 774 SSRs amounting to 91.52 Mb were identified from 131 132 unigenes, with the frequency of 28.81% and the average density of 2.42 kb. Mononucleotide (50.81%) and dinucleotide (34.12%) were the most abundant repeat types. We randomly selected 102 putative loci to test the amplification efficiency and the degree of polymorphism using three populations from which 15 individuals per population were sampled. The successful amplification rate was 36.27% and the polymorphism rate 12.75%. The 13 polymorphic SSR marker primers selected were used to characterize the genetic diversity of M. patungensis. A total of 40 alleles were identified for the species, ranging from 2 to 5 per locus. At the population level, the mean number of alleles per locus ranged from 1.9 to 2.5, and the mean observed and expected heterozygosity values from 0.308 to 0.446 and 0.304 to 0.424, respectively. Significant genetic differentiation (F_ST = 0.286) and low gene flow (N_m = 0.627) among populations were discovered. Transferability of the markers was tested in five congeneric species. Nine loci were successfully amplified in M. dandyi, eight in M. insignis, M. fordiana and M. hookeri, and seven in M. conifera. These new EST-SSR markers will be an essential tool for the conservation of this endangered species as well as for investigating the evolutionary history for M. patungensis and its congenerics.