EST-SSRs were developed in Brassica napus by database mining. We isolated 7,802 EST-SSRs from the B. napus 643,946 ESTs deposited in the NCBI. With the cut-off value of >10 repeats in di-nucleotide repeats and >7 tri-nucleotide repeats, 303 ESTs were suitable for primer designing for PCR amplification. Of the sixteen possible di-nucleotide combinations, only three types of repeats (AC/GT, AG/CT, and AT/TA) were present among the di-nucleotide EST-SSRs. Whereas, 27 tri-nucleotide repeat motifs from the 64 possible combinations were present. The repeat numbers ranged from 10-15 in di-nucleotide repeats and 7-9 in tri-nucleotide repeat motifs, respectively. By checking PCR amplification in 10 Korean rapeseed breeding lines or cultivars, 234 primer pairs showed successful PCR amplification and 142 of the 234 primer pairs revealed polymorphism among the control cultivars or breeding lines. While the repeat length does not related with the SSR polymorphisms, the repeat motif number showed positive relation with the polymorphism generation by showing higher repeat numbers with higher polymorphisms. We are here presenting the PCR amplifiable primer sequences of the 234 SSR primer pairs to aid in the germplasm management and breeding programs of the B. napus in Korea.