To screen the genes related to Brassica napus seed development at pattern formation and maturation stages, the suppression subtractive cDNA libraries of B. napus cultivar Zhongshuang 6 were constructed with its embryos at 10 days after flowering (10 DAF) and 30 days after flowering (30 DAF) through suppression subtractive hybridization (SSH) technology. The positive clones were screened by PCR and dot blot hybridization, and then sequenced. High quality expressed sequence tags (ESTs) were used for COG functional classification with COGNITOR software, as well as analysis and annotation with BLAST software. Tissue-specific detection of five genes screened was performed by RT-PCR in root, stem, leaf, flower, bud, pod, and embryo tissues. The insert size ranged from 100 to 900 bp, with an average size of about 500 bp. According to COG functional classification database, the differentially expressed genes mainly involved in lipid and amino acid metabolism, signal transduction, post-transcriptional modification, and etc. The results from RT-PCR detection of the five differentially expressed genes indicated that genes 2-96 and 2-352 presented embryo-specific expression, gene 1-385 expressed in parts of tissues, and genes 1-71 and 1-682 expressed in all tissues. Two genes were found to be involved in seed development, lipid and protein metabolisms, two genes may be involved in signal transduction, one gene could not match with the homologous sequences known to date and was likely a new gene. These results are helpful for future gene cloning and their functional analysis.