Bovine tuberculosis (BTB) in sheep (Ovis aries) is caused by Mycobacterium bovis and Mycobacterium caprae. Even though sheep have been considered less sensitive to BTB than other ruminants, they have been subject to increasing numbers of tuberculosis cases and it has been suggested that they may act as a disease reservoir in some regions. Aim of the study: Following a report of tuberculous-like gross lesions (repeated cases of purulent or caseous lymphadenitis and a single case of serosal tubercles on the peritoneum) from veterinarians working in a slaughterhouse in the Małopolskie Voivodeship, southern Poland, the aim of this study was to conduct ante-mortem BTB diagnostics in three flocks with suspected BTB. The animals for testing were selected randomly from the flocks; a blood sample for interferon-γ release assay (IGRA) and a single tuberculin skin test (TST) was performed on each sheep. All TST results were negative. The IGRA result was positive in two ewes from the same flock (four and five years of age); these two sheep were tested microbiologically using Stonebrink and Löwenstein-Jensen media. No gross lesions suggesting BTB were observed, and the culture results were negative. Based on the positive IGRA results, together with its high specificity in sheep, and the potential risk to humans posed by consuming local unpasteurized dairy products, we recommend introducing serological BTB screening in sheep from this area, and subjecting the positive results to confirmation by culture.