The current study was carried out to set up a fast and specific technique for porcine tuberculosis diagnosis in formalin-fixed, paraffin-embedded tissues. A retrospective study was carried out using 54 samples fixed in 10% neutral buffered formalin from 29 slaughtered Iberian pigs. Most of the pigs showed tissue samples positive to immunohistochemical staining (70.4%), and mycobacteria were detected within or near the necrotic cores of the lesions. However, diagnosis by this technique was time-consuming and tedious because of the paucibacillar nature of porcine tuberculous lesions. Classic polymerase chain reaction (PCR) was unsuccessful in mycobacteria genome amplification in all of the examined samples; however, real-time PCR amplified the mycobacteria genome in 23 of 29 examined pigs, identifying the Mycobacterium tuberculosis complex in all but one, which amplified Mycobacterium avium complex. Moreover, when reamplification of the DNA was performed, classic PCR amplified the mycobacteria genome in all the examined pigs (29/29), identifying the M. tuberculosis complex in 28 of 29 studied pigs and M. avium complex in only 1 pig. Results of the current study point out that both real-time and classic PCR assays, with genome reamplification, represent sensitive, fast, and specific diagnostic tools for porcine tuberculosis in formalin-fixed, paraffin-embedded tissues.