Johne’s disease is caused by Mycobacterium avium ssp. paratuberculosis (MAP) and has been recognized as an important bacterial infection in ruminants. Although MAP has been detected in semen and within the reproductive organs of bulls, the bacterial distribution and shedding patterns are currently not well characterized. Our investigation was performed to detect and quantify MAP in faeces, semen and blood samples repeatedly drawn from a naturally infected but asymptomatic 18-month-old German Simmental breeding bull candidate over a period of 3 years (June 2007–November 2010). Qualitative and quantitative polymerase chain reaction (PCR) techniques were used to correlate the presence and matrix-specific amounts of MAP. In total, 65 sampling dates were selected. Mycobacterium avium ssp. paratuberculosis was detected intermittently in all matrices with MAP-free intervals of up to 18 weeks by an IS900 semi-nested PCR. The number of MAP-positive results from semen and blood samples was higher than from faecal samples. A quantitative polymerase chain reaction detected the highest MAP contents in faeces (103–106 MAP/g), while lower amounts were found in semen and blood samples (102–105 MAP/ml). Although no significant agreement was calculated between the presence of MAP in faeces and blood, a statistically significant positive correlation between its occurrence in semen and blood was determined (r = 0.38, P < 0.05, n = 29). The present study contributes to a more detailed understanding of MAP distribution patterns in faeces, semen and blood of a subclinically infected breeding bull candidate. It highlights the possible role of breeding bulls as a source of MAP transmission and indicates the need for further monitoring and hygienic measures to prevent the spread of the infection via semen.