Several farms in the Northeast of Brazil were investigated for Mycobacterium avium subsp. paratuberculosis infection in order to identify the occurrence of paratuberculosis in buffaloes. Samples were obtained from 17 farms, two slaughter houses, and a quarantine area in the Northeast. About 15,000 buffaloes of the Murrah, Mediterranean, and Jafarabadi breed as well as their crossbreeds were evaluated for meat, dairy, and mixed farms with semi-intensive or extensive breeding practices. For diagnostic purposes, postmortem and histopathological examination, including Ziehl-Neelsen test of fecal smears and scraped intestinal mucosa were performed. PCR was applied for fecal samples, mesenteric lymph nodes, and intestines. Six Johne's disease-positive farms, which together with those previously identified, indicate that the disease is spread through the Brazilian Northeast, similar to what occurs in cattle herds in other regions of the country. The increase in prevalence of paratuberculosis is a consequence of introduction of animals from other regions without adequate veterinary assistance and due to the little official attention paid to this initially silent and chronic disease.