Abstract Telomeres cap chromosome ends and prevent chromosomal fusions. In the majority of somatic cells telomere shortens with each cell division. Cancer cells, on the other hand, maintain telomere length (TL) through reactivation of telomerase or alternative lengthening of telomere (ALT). Though closely related to cancer hallmarks such as chromosomal instability, telomere length has not been systematically analyzed in cancer. We used DNA sequencing to infer TL in 18,430 samples across 31 cancer types. Tumor TL was shorter compared to normal TL but tended to be longer in testicular germ cell tumors, sarcomas and gliomas. TL in non-neoplastic leukocyte and solid tissue samples was negatively correlated with patient age and varied between lineages, with kidney samples showing the longest TL and leukocytes the shortest. Amongst tumors, 73% expressed telomerase reverse transcriptase (TERT), which was associated with mutations of the TERT promoter (23%), focal amplifications (6%), gene fusions (1%) and structural variants of the promoter (4%) and gene body (3%). Isoform analysis revealed that the full-length TERT transcript was dominantly expressed compared to the enzymatically inactive minus beta variant in all cancer types. TERT expression was positively associated with predicted telomerase activity, inferred from comparison of known ALT and non-ALT tumor samples. Distal breakpoint positions involved in TERT promoter structural variants demonstrated increased levels of H3K27ac and H3K4me1, suggesting displaced enhancer elements. We additionally detected hypermethylation of the TERT promoter in 69%, and found an unexpected association with TERT expression. Combined, 95% of TERT expressing tumors was found positive for at least one potential genomic or epigenetic regulatory event. Six percent of tumors did not express TERT and harbored somatic mutations, deletions, gene fusions or structural variants in ATRX or DAXX, both of which have been shown to be tightly associated with ALT. These tumors demonstrated decreased of ATRX expression in combination with significantly longer TL and expression of telomeric repeat containing RNA (TERRA). Interestingly, 21% of the cohort did not express TERT and was ATRX wild-type. In this double wild-type group, unsupervised analysis identified positive correlations between telomere length, TP53 and RB1 alterations. Predicted telomerase activity in the double wild type and ATRX or DAXX altered groups was significantly lower compared to TERT expressing tumors. Gene expression was found to be TL dependent, with genes nearby telomeres showing a negative correlation (increased expression with shorter TL) whereas genes far away from telomeres showed a positive correlation and (increased expression with longer TL). Our analysis provides insights into the various modalities associated with TERT expression and provides a landscape of determinants of telomere length in cancer. Citation Format: Floris P. Barthel, Siyuan Zheng, Roel G. Verhaak. Comprehensive analysis of telomere length and telomere maintenance mechanisms across 31 human cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3468. doi:10.1158/1538-7445.AM2017-3468