Cellular immortalization is considered a hallmark of cancer and is acquired through the activation of a telomere maintenance mechanism (TMM) that functions to prevent the telomere attrition associated with normal cellular proliferation. It is broadly accepted that cancers employ one of two distinct TMMs: the reverse transcriptase enzyme telomerase (TEL), or a homologous recombination-dependent DNA replication method called alternative lengthening of telomeres (ALT). To further explore the telomere biology of cancer, we characterised TMMs in a panel of 976 cancer-derived cell lines. These analyses involved an initial screen using the C-circle assay (CCA) to detect ALT candidates and a real-time PCR-based assay to quantify telomerase activity. A qPCR measurement was also used to assess telomere content. As expected, the results showed the majority of the cell lines were TEL positive (93%) and a smaller proportion were positive in the C-circle assay (2%). The range of TEL activity and C-circles quantified across the cell line panel was 1000-fold and 400-fold respectively. Notably, in addition to identifying cell lines that were single-positive for either TEL or C-circles, we found subsets of cell lines that were negative in both assays (TMM-double negative), cell lines that were positive for both TEL and C-circles (TMM-double positive) and cell lines with borderline results and ambiguous phenotypes. To validate our initial TMM measures and further investigate novel or ambiguous TMM states, we employed a set of 125 independently prepared cell lines and applied a range of orthogonal assays, including ALT-associated promyelocytic leukaemia body (APB) assay, direct TEL assay, ALT-FISH assay and western blot analysis of ATRX and DAXX (genes that are often mutated in ALT cancers). Telomere length was monitored over time in serially passaged cell lines by terminal restriction fragment (TRF) analysis. These analyses identified cell lines derived from a variety of cancer types that did not maintain their telomere length and lacked the hallmarks of an active TMM as well as cell lines that maintained their telomere length without telomerase or strong phenotypic markers typical of ALT cancers. Collectively, these results demonstrate that TMMs are not binary and highlight the complexity and heterogeneity of cancer telomere biology.