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To track recent thymic emigrants (RTEs) or study T cell development in the thymus, intra-thymic injection of a cellular tag or precursor cells for various T cell lineages is often desired. However, the traditional surgical approach to expose the thymus for intra-thymic injection is time-consuming and can cause a high level of pain and stress to animals, which might disrupt immune homeostasis, potentially confounding the results. Here, we introduce an ultrasound guided intra-thymic injection procedure, which is non-surgical and minimally invasive to animals. This technique is relatively easy to learn and offers an efficient and accurate tool to track RTEs or perform intra-thymic transfer of various cell types to investigate their differentiation., Competing Interests: Competing interestsThe authors declare no competing financial interests., (Copyright © The Authors; exclusive licensee Bio-protocol LLC.)