We report three cases of tuberculosis in alpacas from Spain caused by Mycobacterium bovis. The animals revealed two different lesional patterns. Mycobacterial culture and PCR assay yielded positive results for M. bovis. Molecular typing of the isolates identified spoligotype SB0295 and identical variable-number tandem repeat (VNTR) allele sizes. CASE REPORT Herd 1. The affected herd comprised 32 alpacas (26 adults and six juveniles) and was located in the Ronda region (southern Spain). The animals were raised in outdoor facilities and were fed with commercial pellet feed, hay, and water ad libitum. In March 2009, a 7-year-old female alpaca (alpaca 1) showed dyspnea, fever, depression, lethargy, anorexia, and weight loss. One month later, an 8-year-old male alpaca (alpaca 2) presented chronic weight loss, bruxism, and dyspnea.