Aim: To develop a multiplex real-time PCR assay for the detection and differentiation of Moraxella bovis (M. bovis), M. bovoculi and M. ovis. Methods and Results: The multiplex real-time PCR assay was validated on three reference strains, 57 pure culture isolates and 45 lacrimal swab samples. All reference strains were identified correctly with no cross-reactions between species. Sequencing of 53 of the 57 culture isolates confirmed the results obtained with the multiplex real-time PCR, and the assay had 96.5% (55/57) concordance with a Moraxella spp. multiplex conventional PCR assay on the isolates. Among the lacrimal swab samples, the concordance between the multiplex real-time PCR and culture was 86.7% (39/45) for M. bovoculi and 75.6% (34/45) for M. bovis. Conclusions: The multiplex real-time PCR assay is specific and sensitive and can be used directly on lacrimal swab samples. Significance and Impact of Study: The lack of a rapid, specific and sensitive detection method is a barrier for determining the roles of M. bovis, M. bovoculi and M. ovis in infectious bovine keratoconjunctivitis cases, and the developed PCR assay will contribute to improved understanding of the epidemiology and pathogenesis of these three Moraxella species.