The possible molecular mechanisms regulating sorghum callus regeneration were revealed by RNA-sequencing. Plant callus regeneration has been widely applied in agricultural improvement. Recently, callus regeneration has been successfully applied in the genetic transformation of sorghum by using immature sorghum embryos as explants. However, the mechanism underlying callus regeneration in sorghum is still largely unknown. Here, we describe three types of callus (Callus I-III) with different redifferentiation abilities undergoing distinct induction from immature embryos of the Hiro-1 variety. Compared with nonembryonic Callus III, Callus I produced only some identifiable roots, and embryonic Callus II was sufficient to regenerate whole plants. Genome-wide transcriptome profiles were generated to reveal the underlying mechanisms. The numbers of differentially expressed genes for the three types of callus varied from 5906 to 8029. In accordance with the diverse regeneration abilities observed for different types of callus and leaf tissues, the principal component analysis revealed that the gene expression patterns of Callus I and Callus II were different from those of Callus III and leaves regenerated from Callus II. Notably, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses, pharmacological treatment, and substance content determinations revealed that plant ribosomes, lignin metabolic processes, and metabolism of starch and sucrose were significantly enriched, suggesting that these factors are associated with callus regeneration. These results helped elucidate the molecular regulation of three types of callus with different regeneration abilities in sorghum.