Tertiary lymphoid structures (TLSs) are ectopic lymphoid organs that develop in non-lymphoid tissues, particularly at sites of chronic inflammation and tumors. Key similarities have been identified between the neogenesis of TLSs and the formation of secondary lymphoid organs (SLOs). TLSs can exist in different maturation states, ranging from lymphoid aggregates to fully formed germinal centers. The role of TLSs in the adaptive antitumour immune response is being increasingly understood, with their presence correlated with clinical benefit in cancer patients. Current challenges include leveraging TLSs to enhance lymphocyte infiltration, activation, and differentiation to boost antitumour immune responses. Various approaches, such as using chemokines, cytokines, antibodies, antigen-presenting cells, or synthetic scaffolds, are being explored to induce TLS formation in both immune-low and immune-high tumors. Strategies combining TLS induction with immune checkpoint inhibitors or anti-inflammatory agents show promise for cancer treatment. This review provides a comprehensive analysis of TLS composition, organization, and location in human cancers, focusing on their impact on the tumor microenvironment and antitumour immune responses. It also discusses how TLSs can be used to predict response to therapies, track treatment efficacy, and manipulate antitumour immune responses.Tertiary lymphoid structures (TLSs) are ectopic lymphoid organs that develop in non-lymphoid tissues, particularly at sites of chronic inflammation and tumors. Key similarities have been identified between the neogenesis of TLSs and the formation of secondary lymphoid organs (SLOs). TLSs can exist in different maturation states, ranging from lymphoid aggregates to fully formed germinal centers. The role of TLSs in the adaptive antitumour immune response is being increasingly understood, with their presence correlated with clinical benefit in cancer patients. Current challenges include leveraging TLSs to enhance lymphocyte infiltration, activation, and differentiation to boost antitumour immune responses. Various approaches, such as using chemokines, cytokines, antibodies, antigen-presenting cells, or synthetic scaffolds, are being explored to induce TLS formation in both immune-low and immune-high tumors. Strategies combining TLS induction with immune checkpoint inhibitors or anti-inflammatory agents show promise for cancer treatment. This review provides a comprehensive analysis of TLS composition, organization, and location in human cancers, focusing on their impact on the tumor microenvironment and antitumour immune responses. It also discusses how TLSs can be used to predict response to therapies, track treatment efficacy, and manipulate antitumour immune responses.