Neospora caninum is a cyst-forming coccidian that mainly affects bovines, although Neospora infection has also been described in other domestic and wild ruminant species. Serum samples from 78 alpacas (Vicugna pacos) and 73 llamas (Lama glama) at a unique dilution of 1:50 tested by indirect fluorescent antibody test (IFAT) were further analyzed serologically by IFAT and Western blot in both ruminant species to avoid cross-reactions with closely related coccidian parasites and to confirm the existence of N. caninum–specific antibodies. IFAT titers ranging between 1:50 and 1:800 were found. When using Western blot, N. caninum tachyzoite–specific immunodominant antigens with apparent molecular weights of 17–18, 34–35, 37, and 60–62 kDa were also recognized, although some sera with 1:50 IFAT titers proved not to have N. caninum–specific antibodies. As expected, higher IFAT titers were associated with higher anti–N. caninum reactivity in Western blot. This report documents for the first time the presence of N. caninum infection in adult alpacas and llamas from Peru.