Elicitation of tumor cell killing by CD8+ T cells is an effective therapeutic approach for cancer. A widely utilized strategy to boost anti-tumor immunity is to reinvigorate existing but unresponsive tumor-specific T cells by blocking immune checkpoint receptors such as PD1. Alternative therapeutic approaches also have been developed to overcome the potential limitations of immune checkpoint inhibitory therapy in patients with low pre-existing anti-tumor immunity, including the stimulation of polyclonal T cell cytolytic activity against tumors using Bi-specific T cell engager (BiTE®) antibody constructs that simultaneously engage the T cell receptor (TCR) complex and a tumor-associated antigen. Given that tumour-infiltrating T cells are highly heterogeneous regarding subset composition, gene expression and functional properties, which might contribute to diverse responses to different cancer immunotherapies, an integrated approach, STARTRAC, was developed to utilize TCR sequences as specific markers to quantitatively track the dynamic relationships among T cell subsets identified inside colorectal carcinoma, adjacent normal mucosa and peripheral blood. Using STARTRAC method, we found that there were two distinct activation and expansion routes for tumour-resident effector memory CD8+ Tcells, with one leading to effector CD8+ T cells and the other to exhausted CD8+ T cells, depending on their different TCR usages. These CD8 T cell subsets express distinct checkpoint inhibitors and thus their anti-tumor function might be regulated via distinct pathways. Moreover, our data identified potential novel regulatory molecules such as IGFLR1 for several T cell subsets inside colorectal tumours. These discoveries will shed light on the development of effective immunotherapeutic strategies.