Abstract: In order to develop SSR primers of Cunninghamia lanceolata(Lamb.)Hook based on EST-SSR and genomic SSR, 292 non-repeated Chinese fir EST-sequences were assembled by removing low-quality and redundant fragments depend upon 444 ESTs sequences from NCBI Public database, and 143 genome-sequences were assembled by removing redundant fragments in 1 142 genome sequences. All those non-redundant sequences of Cunninghamia lanceolata(Lamb.)Hook was searched for mono-to hexa-nucleotide simple sequence repeats(SSR or microsatellite)with software MISA, and the type,size and frequency of these SSRs were determined. Totally, There were 109 SSRs to be picked out among EST-sequences, the distribution density was 964.58 SSR/Mbp; and 39 SSRs were found in genome sequences, accounting for the higher density of 1 037.24 SSR/Mbp. In both Genomic and EST sequence library, the Hexanucleotides repeats, especially AT-rich repeat, was the most repeated type, and the AGC/CTG was the most trinucleotides repeats, The 37 and 95 pairs of SSR primers were designed based on EST library and Genomic by Primer 3.0, respectively. Ten EST-SSR primers and eight pairs of gSSR primers were selected randomly to perform PCR amplification with 12 individuals. There were four pairs of primers showed polymorphism both in EST-SSR and gSSR, with polymorphic rate of 40% and 50%, respectively. Twenty-five polymorphic alleles were amplified by 8 SSR primer pairs, which showed an average 3.125 polymorphism alleles for each primer, the average effective alleles(Na)2.399 5, average PIC 0.519 1, average Hot 0.307 4. There were more polymorphism alleles from gSSR markers than EST-SSR among groups. More allelic loci were amplified with four pairs of primer from gSSR than the four pairs of primer from EST-SSR, and the former had higher PIC value also.