Oncidium 'Gower Ramsey' is a valuable and successful commercial orchid for the floriculture industry in Taiwan. However, no genome reference sequence currently exists for Oncidium orchids, to facilitate the development of molecular biological studies and the breeding of these orchids. In this study, we generated Oncidium cDNA libraries for six different organs, including leaves, pseudobulbs, young inflorescences, inflorescences, flower buds and mature flowers. We utilized 454-pyrosequencing technology to perform high-throughput deep sequencing of the Oncidium transcriptome, yielding more than 0.9 million reads with an average length of 328 bp, for a total of 301 million bases. De novo assembly of the sequences yielded 50,908 contig sequences with an average length of 493 bp from 796,463 reads and 120,219 singletons. The assembled sequences were annotated using BLAST, and a total of 12,757 and 13,931 UniGene transcripts from the Arabidopsis and rice genomes were matched by TBLASTX, respectively. A Gene Ontology (GO) analysis of the annotated Oncidium contigs revealed that the majority of sequenced genes were associated with cellular processes, unknown molecular functions and intracellular components. Furthermore, a complete flowering-associated expressed sequence that included most of the genes in photoperiod pathway and the 15 CONSTANS-LIKE (COL) homologs with the conserved CCT domain was obtained in this collection. These data revealed that the Oncidium EST database generated in this study has sufficient coverage to be used as a tool to investigate the flowering pathway and various other biological pathways in orchids.