Approximately 10% of cancers overall use alternative lengthening of telomeres (ALT) instead of telomerase to prevent telomere shortening, and ALT is especially common in astrocytomas and various types of sarcomas. In this regard, easy and accurate detection of ALT in tumour cells is important both for clinical applications and cancer biology research. The hallmarks of ALT in telomerase-negative cancer cells include a characteristic pattern of telomere length heterogeneity, rapid changes in individual telomere lengths and the presence of ALT-associated PML bodies (APBs) containing telomeric DNA and proteins involved in telomere binding, DNA replication and recombination. The methods currently used for analysing these phenotypes include Southern-based terminal restriction fragment (TRF) analysis, APB-staining and fluorescence in situ hybridisation (FISH)-based telomere length fluctuation analysis. Details of these three methods, together with a screen for candidate ALT genes, are described in this review.