Tissue culture is a crucial step in the genetic transformation of peanut（Arachis hypogaea L. ）. However， the differentiation of peanut callus is restricted by genotype. In this study， Luohua 22， a cultivar with strong callus differentiation ability， was screened from 9 germplasm resources through establishing a peanut genetic transformation system. Transcription sequencing analysis were carried out using T1 callus（embryogenic callus prone to differentiate into seedlings）， T2 callus（non-embryogenic callus difficult to differentiate into seedlings）during the differentiation process of Luohua 22， and callus at 0 day of differentiation was set as control（CK）. Compared with CK， there were 1792 and 868 differentially expressed genes（DEGs）in T1 and T2 types of callus， respectively. The GO term enrichment analysis showed that the DEGs in T1 type of callus were mainly enriched in the terms of meristematic organization and stem cell population maintenance， while those in T2 type of callus were mainly enriched in the terms of phenylpropane biosynthesis and metabolic term. Protein family analysis showed that DEGs in T1 and T2 types of callus contained 2459 protein-coding genes， among which the cytochrome P450 family was significantly enriched. By protein-protein interaction（PPI）network analysis， five key genes AhAE3ZZG， AhP17M1H， AhA6R79F， AhZFZ3ZQ and AhHMN99B， were mined， which might play important roles in promoting peanut callus differentiation. The results of this study would provide a scientific basis for furhter exploring the key genes affecting the differentiation process of peanut cotyledon node callus， and the subsequent analysis of the molecular mechanism of peanut callus differentiation to form regenerated plants.