The root of Gentiana macrophylla is a traditional Chinese medicine that has been used for over 2000 years. We introduced seed priming to improve germination performance for this species and found that after ferrous sulphate priming (FeP), seeds presented higher germination under both optimal and adverse environments than non-primed (NP) seeds. Transcriptome analysis conducted on FeP, hydro-primed (HP) and NP seeds was used to identify the key genes involved in metabolism and contribute to understanding of the molecular mechanisms of seed priming. After calculating the gene expression and analysing the differentially-expressed genes (DEGs), a total of 2050 and 914 unigenes were identified as differentially expressed in FeP and HP seeds, respectively, relative to NP seeds. On the basis of the KEGG pathway enrichment analysis results, by analysing and summarising those DEGs related to enzymes during metabolism, enzymes involved in carbohydrate, nucleic acid metabolism, hormone biosynthesis and signal transduction, respiration and energy metabolism, and stress-related metabolism were all regulated after FeP treatment. Their regulated trends exhibited almost similar patterns with the initial germination process, whereas those from HP treatment were weaker than the effect of FeP, where only a small number of enzymes were regulated. Primed seeds are developmentally more advanced towards the completion of germination than NP seeds.