The VP2 protein of bluetongue virus (BTV) is an important structural protein and is the principal antigen responsible for BTV serotype specificity. In this study, we mapped the reactivity of two BTV16-specific monoclonal antibodies (MAbs) and identified two novel serotype-specific linear B cell epitopes on the BTV16 VP2 protein. By screening a series of peptides derived from the BTV16 VP2 protein and expressed as mannose-binding protein fusions, we determined that the linear epitopes recognized by the VP2-specific MAbs 3 G10 and 2B4 were located within the peptides (34)EWSGHDVTEIPNRRMF(49) and (540)KNEDPYVKRTVKPIRA(555), respectively. To define the minimal region required for antibody binding within these peptide regions, a series of progressively shorter peptides were synthesized and evaluated for 3 G10 and 2B4 binding. This work defined the motifs (34)EWSGHDVTEIPNRRMF(49) and 543DPYVKRTVK555 as the minimal linear peptides required for 3 G10 and 2B4 binding, respectively. Alignment of amino acid sequences from a number of BTV16 strains isolated from different regions indicated that these two epitopes are highly conserved among BTV16 strains. Furthermore, these two epitopes are not conserved among other BTV serotypes or prototype members of the genus Orbivirus in the family Reoviridae, as shown by sequence alignments. The MAb reagents and linear epitopes defined here provide the basis for the development of epitope-based serotype-specific differential diagnostic tools and may be useful in the design of epitope-based vaccines.