Bitter gourd, Momordica charantia L., is an economically and nutraceutically important crop in the family Cucurbitaceae. The predominant sex-form of bitter gourd is monoecious, but a gynoecious form also exists which is controlled by a single recessive gene (gy-1). Whole transcriptome sequencing of female and hermaphrodite flower buds of bitter gourd was performed using Roche 454 parallel pyrosequencing technology. A total of 1,994,113 expressed sequence tags (ESTs) were generated, of which 834,476 and 1,159,637 ESTs were obtained from female and hermaphrodite flower buds of the gynoecious bitter gourd inbred line, DBGY-201, respectively. The unigenes were functionally annotated by comparing their sequences with various protein and functional domain databases and assigned gene ontology (GO) terms. A total of 477 annotated unigenes were found to be significantly differentially expressed between the female and hermaphrodite flower buds of DBGY-201, of which 237 were down-regulated and 59 were up-regulated in hermaphrodite flower buds. Differentially expressed ESTs between the two different flower types, as well as putative simple sequence repeat (SSR) markers are reported. These EST sequences will provide valuable information to increase our understanding of the molecular mechanisms of sex determination and represent a rich resource for future functional genomic analysis, marker development, and bitter gourd breeding.