Effective vaccination induces memory T cells, which protect the host against pathogen re-infections. Therefore, detection of memory T cells is essential for evaluating vaccine efficacy, which was originally dependent on cytokine induction assays. Currently, two isoforms of CD45 tyrosine phosphatase, CD45RO expression and CD45RA exclusion (CD45RO+/ CD45RA-) are used extensively for detecting memory T cells in cattle. The CD45RO+/CD45RA- markers were first established in humans around three decades ago, and were adopted in cattle soon after. However, in the last two decades, some published data in humans have challenged the initial paradigm, and required multiple markers for identifying memory T cells. On the contrary, memory T cell detection in cattle still mostly relies on CD45RO+/CD45RA- despite some controversial evidence. In this review, we summarized the current literature to examine if CD45RO+/CD45RA- are valid markers for detecting memory T cells in cattle. It seems CD45RA and CD45RO (CD45RA/RO) as markers for identifying bovine memory T cells are questionable.