Peanut (Arachis hypogaea L.) is the fourth largest oil crop in the world. Tissue culture is an important procedure in genetic transformation of peanut. 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 analyses were carried out using T1 callus (embryogenic callus, easy 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 functional enrichment analysis showed that the DEGs in T1 type of callus were mainly enriched in the pathways of meristematic organization and stem cell population maintenance, while those in T2 type of callus were mainly enriched in the pathways of phenylpropane biosynthesis and metabolic pathway. Protein family analysis showed that DEGs in T1 and T2 type callus contained 2459 TFs, among which the cytochrome P450 family was significantly enriched. By Protein-protein interaction network (PPI) analysis, five key genes, i.e., 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.