An alpaca (Vicugna pacos) bred and kept in a German zoological garden exhibited clinical signs consistent with paratuberculosis. The presence of Mycobacterium avium ssp. paratuberculosis (MAP) was confirmed in feces and in the ileocecal lymph node (ILN) by IS900-based polymerase chain reaction (PCR) assays and culture. A bacterial burden of 7.6 × 10⁶ MAP/g in feces and 4.4 × 10⁷ MAP/g in lymph node tissue was determined by real-time PCR. For further characterization, a conventional PCR was developed. After sequencing of the 864-bp PCR amplicon covering nucleotide positions 13 to 876 within the IS900, the alpaca isolate shared 100% nucleotide homology with the bovine MAP-K10 IS900 reference sequence (GenBank: AE16958), indicating a cattle strain. This report supports the present occurrence of MAP in German camelid populations and highlights the need to expand routine MAP surveillance to South American camelids held in European zoos.