Peach is a popular and economically important fruit tree highly susceptible to biotic and abiotic stresses, including diseases caused by fungi, bacteria and viruses, pests and insects that feed fruits and leaves. Alteranria is a deadly disease agent, a genus of fungi that can cause significant damage to plants and crops such as peaches. Here, we investigated the role of CPK genes against in Alternaria species. The calcium-dependent protein kinase (CPK) gene family plays a vital role in the biotic stress response of plants. In this study, we aimed to identify, characterize, and analyze the expression pattern of the PpCPK gene family in response to Alternaria-infected peach fruits collected from different geographical regions of China. Based on phylogenetic analysis, 17 PpCPK genes were identified and classified into seven groups. The physicochemical properties, gene structure, conserved domains, and motifs of the PpCPK genes were analyzed to understand their molecular characteristics further. The Ka/Ks ratio analysis revealed the evolutionary facets of duplication events of PpCPKs and predicted the divergence time between 23.5 and 43.6 million years ago. The promoter region analysis confirmed the presence of plant growth, light, stress, and hormone-related cis-elements in PpCPKs. Additionally, the expression pattern of PpCPKs was analyzed using real-time quantitative PCR in Alternaria-infected peach fruits from different regions of China. The results indicated a differential expression pattern of PpCPK genes responding to Alternaria infection, suggesting their potential role in defense against biotic stress. Overall, our study provides insights into the molecular mechanisms of PpCPK genes in response to biotic stress, which could aid in developing strategies for improving peach resistance to biotic stress.