Glycyrrhiza is one of the important medicinal plants that is in danger of extinction. Search for finding accessions that have a higher glycyrrhizic acid is very important in breeding programs. Functional genomics methods such as EST sequencing prepare the ability to identify consensus gene families among studied species and interpretation of the genome. In this research, 55960 EST sequences of two different species of this plant along with the protein sequences were analyzed in order to identify the molecular aspects and functional analysis of the genome and the gene network involved in the biosynthesis of glycyrrhizin. Also, in order to validation of results, relative expression of four important genes in the pathway of glycyrrhizin biosynthesis including squalene synthase (SQS), β-amyrin synthase (BAS), β –amyrin 11-oxidase (CYP88D6) and UDP-glucuronyl transferase (UGT) were evaluated. After trimming and qualitative evaluation the sequences, 6427 contig sequences and 30895 singleton (37322 unigene) were generated, which covered a total of 26884666 bp (7.06%) of the licorice genome. Genome functional activity showed that most genes play a role in the catalytic activity and cellular and metabolic processes in which these genes interact within cells and intracellular organels. Locating this group of genes showed that the genes involved in glycyrrhizin biosynthesis pathway were localized in endoplasmic reticulum. Results of validation using qRT-PCR showed that in the autumn and in the rhizome tissue, the genes of BAS, CYP88D6, UGT and SQS were up-regulated. The results of this study can be valuable for genomic sequencing, functional groups, genetic diversity and functional genomics of this plant.