The gynoecium is one of the most complex organs of angiosperms specialized for seed production and dispersal, but only several genes important for ovule or embryo sac development were identified by using female sterile mutants. The female sterility in oilseed rape (Brassica napus) was before found to be related with one alien chromosome from another crucifer Orychophragmus violaceus. Herein, the developmental anatomy and comparative transcript profiling (RNA-seq) for the female sterility were performed to reveal the genes and possible metabolic pathways behind the formation of the damaged gynoecium. Using Brassica_ 95k_ unigene as the reference genome, a total of 28,065 and 27,653 unigenes were identified to be transcribed in S1 and H3, respectively, suggesting the newly initiated transcriptions in S1. Further comparison of the transcript abundance between S1 and H3 revealed that 4540 unigenes showed more than two fold expression difference. Gene ontology and pathway enrichment analysis of the Differentially Expressed Genes (DEGs) revealed that a number of important genes and metabolism pathways were involved in the development of gynoecium, embryo sac, ovule, integuments as well as the interactions between pollen and pistil. DEGs for the ovule development were detected to function in the metabolism pathways regulating brassinosteroid (BR) biosynthesis, adaxial/abaxial axis specification, auxin transport and signaling. A model was proposed to show the possible roles and interactions of these pathways for the sterile gynoecium development. The results provided new information for the molecular mechanisms behind the gynoecium development at early stage in B. napus. Overall design: Gynoecium samples in 1.5 to 3mm long flower buds were collected from Brassica napus (H3) and its female sterile addition line (S1). Total RNA from the two samples were extracted and sequencing was performed using Illumina HiSeqâ„¢ 2000.