Tuberculosis has been diagnosed in wild boar (Sus scrofa) in several European countries during the last decade; however, almost no information has been reported to date for Portugal. This study aimed to investigate tuberculosis in wild boar in Portugal through characterization of Mycobacterium bovis infection and identification of disease risk factors. Tissue samples were obtained from hunted wild boar during the 2005 and 2006 hunting seasons. Samples were inspected for gross lesions and processed for culture. Acid-fast bacterial isolates were identified by polymerase chain reaction and spoligotyping. Associations between tuberculosis in wild boar and several variables linked to wild ungulate diversity and relative abundance, livestock density, and cattle tuberculosis incidence were investigated. Mycobacterium bovis isolates were identified in 18 of 162 wild boars from three of eight study areas. Infection rates ranged from 6% (95% confidence interval [CI(P95%)] = 1-21%) to 46% (CI(P95%) = 27-67%) in the three infected study areas; females in our sample were at greater risk of being infected than males (odds ratio = 4.33; CI(P95%) = 3.31-5.68). Spoligotyping grouped the M. bovis isolates in three clusters and one isolate was a novel spoligotype not previously reported in international databases. Detection of M. bovis was most consistently associated with variables linked to wild ungulate relative abundance, suggesting that these species, particularly the wild boar, might act as maintenance hosts in Portugal.