African swine fever, which is induced by the African swine fever virus (ASFV), poses a significant threat to the global pig industry due to its high lethality in domestic pigs and wild boars. Despite the severity of the disease, there is a lack of effective vaccines and drugs against the ASFV. The p72 protein, constituting 31 to 33% of the total virus particle mass, serves as the primary capsid protein of ASFV. It is a crucial antigen for the development of ASF subunit vaccines and serological diagnostic methods. In this investigation, 27 monoclonal antibodies (mAbs) were generated through mouse immunization with the truncated C-terminal p72 protein expressed by Escherichia coli. Among these, six mAbs exhibited binding to the p72 trimer, with their respective recognized epitopes identified as ⁵⁴²VTAHGINLIDKF⁵⁵³, ⁵⁶⁸GNAIKTP⁵⁷⁴, and ⁵⁸⁴FALKPREEY⁵⁹². All three epitopes were situated within the interval sequences of functional units of the C-terminal jelly-roll barrel of p72. Notably, two epitopes, ⁵⁶⁸GNAIKTP⁵⁷⁴ and ⁵⁸⁴FALKPREEY⁵⁹², were internal to the p72 trimer, while the epitope ⁵⁴²VTAHGINLIDKF⁵⁵³ was exposed on the surface of the trimer and consistently conserved across all ASFV genotypes. These findings enhance our comprehension of the antigenic function and structure of the p72 protein, facilitating the utilization of p72 in the development of diagnostic techniques for ASFV.