Four commercial ELISA kits for bovine paratuberculosis were evaluated using individual serum samples from 53 dairy cattle in 10 infected dairy herds and 368 dairy cattle in 11 paratuberculosis-free herds of southern Chile. Blood samples were collected in 10 ml Vacutainer tubes and the sera frozen at -20°C until tested in duplicate following manufacturer's recommendations. The infection status of the infected herds was determined by fecal culture on a single sampling and for the paratuberculosis-free herds by two consecutive 100% negative fecal cultures one year apart. Fecal samples were collected via rectum using individual polyethylene sleeves, transported to the lab, and cultured within 24h on home-made Herrold's Egg Yolk Medium (HEYM) with mycobactin J (3 tubes) and HEYM without mycobactin J (1 tube), using the centrifugation method. Prior to culture, 2g of each fecal sample was decontaminated with HPC and an antibiotic solution containing nalidixic acid, vancomycin, and amphotericin B. A 0.15 ml aliquot of each suspension was used to inoculate all HEYM tubes which were incubated at 37°C for 16 weeks. Colonies resembling M. paratuberculosis and showing mycobactin-dependence were tested and confirmed by IS900 PCR technology. The following cutoff values recommended by the manufacturers were used for sensitivity and specificity analysis of each kit: S/P ≥ 0.25 (kit A), S/P% ≥ 70 (kit B), (OD-neg) ≥ 0.100 (kit C), and S/P% ≥ 70 (kit D). The specificity of the four ELISAs were 99.73% (kit A), 99.18% (kit B), 99.46% (kit C), and 98.76% (kit D). The sensitivity of the four kits for detecting fecal culture-positive cows were: 32.08% (kit A), 37.74% (kit B), 41.51% (kit C), and 37.74% (kit D). Receiver Operating Characteristic (ROC) analysis showed that kit C performed much better than the other three kits as the AUC values for the four kits assayed were 0.899 (kit A), 0.818 (kit B), 0.945 (kit C), and 0.854 (kit D). Assay agreement between kits was high (kappa 0.842 to 0.908) for categorical interpretations (positive or negative); the following kappa values were calculated for all four kits: A vs B = 0.843; A vs C = 0.843; A vs D = 0.842; B vs C = 0.908; B vs D = 0.907; and C vs D = 0.952. According to these results all four paratuberculosis ELISA kits evaluated were similar in sensitivity and specificity but kit C was most accurate.