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Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis (PTB), disease that causes a syndrome of bad nutrient absorption, weight loss and eventually death. The intestine is the main target organ where the infection develops; however, there is evidence of infection by MAP in extra-intestine sites of sheep, including mesenteric nodes and semen. The aim of the study was to identify the presence of MAP in reproductive tissue and semen of infected Pelibuey rams in clinical state of PTB. Seven rams were used in clinical PTB state and a non-infected ram by MAP of the Pelibuey breed, confirmed by serology, nPCR and bacteriological culture, with average weight and age of 57.23 ± 1.73 kg and 2.91 ± 0.17 years, respectively. The presence of MAP was identified in different tissue samples: spleen (1/7, 14.3% and 2/7, 28.6%), small intestine (3/7, 42.9% and 4/7, 57.1%) and mesenteric lymph nodes (3/7, 42.9% and 3/7, 42.9%), with nPCR and culture, respectively. It was also identified in epididymis tissue (1/7, 14.3%), Cowper gland (2/7, 28.6%) and prostate (1/7, 14.3%), using nPCR, although without detection in culture. It was identified in testicular tissue in 42.8% (3/7; culture or nPCR technique), but in 28.6% (2/7) with both techniques. Finally, the presence of MAP was identified in 42.9% (3/7) of semen samples with nPCR; however, it was not detected through culture. In conclusion, the presence of MAP was identified in lymphatic, digestive tissue, and semen; the presence of MAP was reported for the first time in epididymis, Cowper gland, prostate and testicles of infected Pelibuey rams.