Cytochrome P450 (CYP) genes encode enzymes that catalyze various growth-, development-, and stress-related reactions. Sorghum (Sorghum bicolor) is a type of C4 plant and an important cash crop. However, systematic identification and analysis of functional differentiation and evolution of CYP genes have not been carried out in this species. In the present study, we revealed that the sorghum genome contains 351 CYP genes, which can be divided into nine classes. These genes are from ancestors and repeated segments, rather than tandem repeats. Based on collinearity results, a large number of CYPs were extended before cotyledon differentiation, during the emergence of Gramineae, suggesting that genomewide duplication events and stress adaptation processes were important for the expansion of CYP genes. Their gene structure and motifs contain conserved regions and include various changes and loci. The expression characteristics and functional annotation of CYP genes indicated tissue specificity and selective expression. Overall, we identified all CYP genes in the sorghum genome and preliminarily explored their naming, structure, evolution, expression, and functional differentiation. The results advanced our understanding of plant gene family evolution and functional differentiation.