Mammalian chromosome ends are protected by nucleoprotein structures called telomeres. Cancer cells must activate a telomere maintenance mechanism to achieve proliferative immortality. The majority of cancer cells activate the ribonucleoprotein enzyme telomerase, while a smaller proportion engages the Alternative Lengthening of Telomeres (ALT) pathway. ALT is prevalent in aggressive tumour subtypes, such as sarcomas and astrocytomas; however, the underlying mechanism of ALT-mediated telomere elongation remains unknown. We have utilised telomere fibre-fluorescence in situ hybridisation (fibre-FISH) to visualise telomeric recombination and replication events in ALT cells. By employing a constant stretching factor of 2 kb/μm to stretch DNA fibres, followed by hybridisation to a telomere-specific PNA probe, we have measured telomere length in ALT cancer cells stably overexpressing either the Holliday junction (HJ) dissolvase BLM or the HJ resolvase SLX4. We identified telomere lengthening in BLM overexpressing ALT cell lines, and telomere shortening in SLX4 overexpressing ALT cell lines. We then adapted the single molecule analysis of replicated DNA (SMARD) technique for the specific visualisation of telomere extension events. Single molecule analysis of telomeres (SMAT) revealed that BLM is required for ALT-mediated telomere synthesis, while overexpression of SLX4 reduced the frequency of telomere extension events. Overall, our data demonstrate that BLM is required for telomere synthesis in ALT cell lines, and that this process is counteracted by SLX4. Telomere analysis by fibre-FISH can be used to accurately measure telomere length, as well as telomere replication and synthesis dynamics at the single molecule level.