Telomeres are terminal repetitive DNA sequences at the ends of chromosomes, and are considered to consist almost exclusively of the hexameric sequence TTAGGG. We analysed telomeres in humans using whole-genome sequencing followed by telomeric read extraction in a panel of mortal and immortal cell lines. We identified a wide range of telomere variant repeat sequences in human cells, and found evidence that telomerase- and ALT-mediated telomere lengthening generate variant repeats in mechanistically distinct ways. Telomerase-mediated telomere extension resulted in the generation of biased variant repeats that differed to the canonical sequence at positions 1 and 3, but not at positions 2, 4, 5 or 6. In contrast, cell lines that use the ALT pathway of telomere maintenance contained a large variety of variant repeats that differed between lines. This is consistent with variant repeats spreading from proximal telomeric regions throughout telomeres in a stochastic manner by recombination-mediated templating of DNA synthesis. The presence of unexpectedly large numbers of variant repeats in cells utilizing either telomere maintenance mechanism suggests a conserved role for variant sequences at human telomeres. To further investigate this we have carried out a comparative evaluation of telomere sequence content in different organisms and found that telomere variant repeat profiles differ between species. We propose that different variant repeats fulfil specific functional roles within telomeres, and are currently investigating the mechanisms underlying variant repeat generation.