Crimean-Congo hemorrhagic fever (CCHF), a severe viral disease known to have occurred in over 30 countries and distinct regions, is caused by the tick-borne CCHF virus (CCHFV). Nucleocapsid protein (NP), which is encoded by the S gene, is the primary antigen detectable in infected cells. The goal of the present study was to map the minimal motifs of B-cell epitopes (BCEs) on NP. Five precise BCEs (E1, 247FDEAKK252; E2a, 254VEAL257; E2b, 258NGYLNKH264; E3, 267EVDKA271; and E4, 274DSMITN279) identified through the use of rabbit antiserum, and one BCE (E5, 258NGYL261) recognized using a mouse monoclonal antibody, were confirmed to be within the central region of NP and were partially represented among the predicted epitopes. Notably, the five BCEs identified using the rabbit sera were able to react with positive serum mixtures from five sheep which had been infected naturally with CCHFV. The multiple sequence alignment (MSA) revealed high conservation of the identified BCEs among ten CCHFV strains from different areas. Interestingly, the identified BCEs with only one residue variation can apparently be recognized by the positive sera of sheep naturally infected with CCHFV. Computer-generated three-dimensional structural models indicated that all the antigenic motifs are located on the surface of the NP stalk domain. This report represents the first identification and mapping of the minimal BCEs of CCHFV-NP along with an analysis of their primary and structural properties. Our identification of the minimal linear BCEs of CCHFV-NP may provide fundamental data for developing rapid diagnostic reagents and illuminating the pathogenic mechanism of CCHFV.