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The present study highlights the transboundary nature of tuberculosis (TB) in alpacas and the failure of current antemortem testing protocols to identify TB‐free alpaca herds and individuals for exportation. The tuberculin skin test (TST) failed to identify Mycobacterium bovis‐infected animals prior to movement from the United Kingdom (UK) to Poland. This study describes the use of four serological assays [Enferplex Camelid TB, dual‐path platform (DPP) VetTB and BovidTB assays, and multi‐antigen print immunoassays (MAPIAs)] to detect TB in an alpaca herd with negative TST results. The breeding in Poland purchased alpacas for several years from the UK with the last group arriving in May 2018. In July 2018, two sick alpacas from the centre were hospitalized in a veterinary clinic and both died of TB a few weeks later. In November 2018, 20 alpacas remaining in this M. bovis‐affected herd were euthanized and samples were collected. The study population included 20 M. bovis‐infected and 20 uninfected alpacas, but only 15 infected animals were tested by all serology tests. The DPP VetTB and DPP BovidTB assays detected antibodies in 14 of the 20 infected alpacas, with results confirmed by MAPIA, and in none (MAPIA and DPP BovidTB) or one (DPP VetTB) of the 20 uninfected animals. None of the infected alpacas tested positive using the Enferplex assay. In addition, the group included three orphans and two cria–dam pairs, which provided an opportunity to analyse immune aspects of cria–mother relationships in this herd. The results suggest high susceptibility of this host species to M. bovis infection and rapid progression to disease. The serological tests used in this study offer useful tools for the detection of M. bovis infection in TST and Enferplex test non‐reactive alpacas. These tests should be further evaluated for implementation into TB management and control strategies for camelid species. [ABSTRACT FROM AUTHOR]