From the end of 2004 increasing numbers of tuberculosis-like lesions were detected in regular meat inspections of pigs at regional abattoirs. Mycobacterium avium subspecies hominissuis was identified as the cause of this outbreak of tuberculosis lymphadenitis by IS901 and IS1245 duplex PCR and FR300 PCR. In order to detect the source and route of infection, IS1245 restriction fragment length polymorphism (RFLP) analysis was used to type 54 strains (50 from pigs, one from a cow, two from wild boar and one from peat) isolated in 12 different regions of Portugal. A total of 51 different patterns were obtained with most strains giving a high IS1245 copy number (between seven and 27) with a complex RFLP pattern. Twenty-three strains were gathered in 11 clusters, defined as having at least 80 per cent similarity. Identical isolates were obtained from one herd in Lisbon (two isolates), one herd in Setúbal (two isolates) and in two isolates from different herds in the same region. No distinctive geographical clustering was found among the 54 strains. These results indicate that strains of M hominissuis are very heterogeneous and widespread in pigs in Portugal. No major common source of the infection was identified.