Mucosal surfaces such as the gastrointestinal tract, and skin are the front line of host defence and immunity against many pathogens. Gamma delta (γδ) T lymphocytes preferentially localize to the mucosal surfaces in several species including cattle, and are thought to play crucial roles in immunosurveillance and host defence, particularly against mycobacteria. Many γδ T cells are present in young calves, which is the period when calves are thought to be initially exposed to Mycobacterium avium subspecies paratuberculosis (Map). The role of mucosal γδ T cells in cattle, especially during host-pathogen interactions during early pre-clinical phases of infectious disease remains unclear. The purposes of this study were to investigate and characterize WC1+ and WC1neg γδ T cell subsets in various segments of the gastrointestinal (GI) tract of young calves, and then to examine γδ T cell subsets in the distal small intestine of calves after experimental intestinal Map infection by direct Peyer's patch inoculation. We show that in healthy calves, the relative proportion of γδ T cells is constant throughout the GI mucosa, though the ileum has significantly more γδ T cells. In the distal intestine, γδ T cells are mainly WC1neg and primarily located within the lamina propria of the jejunum and ileum. In Map-infected intestine, there are higher numbers of γδ T cells in the lamina propria and a greater proportion of WC1+ cells within the epithelial layer compared to control calves. While WC1neg γδ T cells preferentially localize to the distal small intestine of healthy calves, WC1+ γδ T cells are increased in the intestinal mucosa during Map infection, which is suggestive of effector cell function. Further, spectral microscopy and flow cytometry in tandem will lead to improved understanding of the functions of these cells during health and disease. Copyright © 2019 Elsevier B.V. All rights reserved.