Agaricus subrufescens is one of the most important culinary-medicinal cultivable mushrooms with potentially high added-value products and extended agronomical valorization. The development of A. subrufescens related technologies is hampered by, among others, the lack of suitable molecular tools. Thus, this mushroom is considered as a genomic orphan species with a very limited number of available molecular markers or sequences. To fill this gap, this study reports the generation and analysis of the first set of expressed sequence tags (EST) for A. subrufescens. cDNA fragments obtained from young sporophores (SP) and vegetative mycelium in liquid culture (CL) were sequenced using 454 pyrosequencing technology. After assembly process, 4989 and 5125 sequences were obtained in SP and CL libraries respectively. About 87 % of the EST had significant similarity with Agaricus bisporus 36 predicted proteins and 79% correspond to known proteins. Functional categorization according to Gene Ontology could be assigned to 49 % of the sequences. Some gene families potentially involved in bioactive compounds biosynthesis could be identify. A total of 232 simple sequence repeats (SSR) were identified and a set of 40 EST-SSR polymorphic markers were successfully developed. This EST dataset provides a new resource for gene discovery molecular markers development. It constitutes a solid basis for further genetic and genomic studies in A. subrufescens.