Abstract Genome-wide DNA sequence resources are expected to enhance our understanding of the molecular basis of plant development and lead to improvements in desirable traits in horticultural crops. In this study, we sequenced 643,366 ESTs from Eustoma grandiflorum flowers using a normalised cDNA library constructed for several different flower development stages, times of day, and pollinated stamens. The sequences were assembled into 63,401 contigs and 242,212 singletons. BlastX searches for all of the contigs in the GenBank database matched 65% of the contigs to registered sequences, while 35% presented no hits. GO mapping assigned 48% of the 63,401 contigs to GO terms. Microarray analysis showed that subsets of genes were up- or downregulated as the flower developed. The upregulated genes were enriched for GO terms related to (1) oligopeptide transport, (2) response to jasmonic acid stimulus, and (3) cell wall modification, whereas the downregulated genes were enriched for GO terms involved in (1) secondary metabolism, such as the flavonoid biosynthetic process and the terpenoid biosynthetic process, and (2) epidermal cell modification.