Simple sequence repeats (SSRs) are important components of eukaryotic genomes and may play an important role in regulating gene expression. However, the characteristics of genic SSRs and the effect of interspecific hybridization and polyploidization on genic SSRs seem not to have received desired attention in terms of scientific investigations. To determine the features of genic SSRs and elucidate their role in polyploidization process of the Brassica family, we identified SSRs in Plant Genome Database-assembled unique transcripts (PUTs) of Brassica species. A higher density of SSRs and a greater number of compound motif SSRs and mononucleotide motif types with large average number of repeats were detected in allotetraploid Brassica napus than in the diploid parental species (Brassica rapa and Brassica oleracea). In addition, a greater proportion of SSR-PUTs were found to be associated with the stress response and developmental processes in B. napus than in the parents. A negative correlation between the repeat number and the motif type and the total length, and a positive correlation between the repeat number and the total length of SSRs were observed. PUT-SSR might be generated from A/T-rich regions. The successful development of 123 pairs of SSR primers for Brassica PUTs showed that SSR-PUTs could be exploited as gene-based SSR functional markers for application in Brassica breeding. These results indicate that interspecific hybridization and polyploidization could trigger the amplification of SSRs, and long SSRs might become shorter to enable the plant to adapt to environmental and artificial selection.