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Sweet-type cultivars of cassava (Manihot esculenta Crantz) are consumed boiled directly after harvest. Farmers are selecting landraces with various levels of sugars, and the characterisation of germplasm collections necessitates robust and accurate techniques for high-throughput analysis of numerous accessions. The objectives of the present study were to develop and test a high performance thin layer chromatography (HPTLC) protocol for the fast quantitation of the cyanogenic glycoside linamarin and individual sugars (maltose, sucrose, glucose, fructose), to determine variation within and between roots and different clones of the same genotype, as well as to compare 147 landraces and 65 hybrids from Vanuatu for their total sugar contents. There is variation in total sugar content in the fresh root depending on where the sample is collected. A protocol based on the analysis of three samples per root (proximal, central and distal parts) was used to compare roots of the same plant and clones of the same accession. Overall, 1030 samples were analysed for major and unidentified minor sugars. For the 147 landraces, the minimum sugar content was 2.53 g (per cent fresh weight) and the maximum 10.36 g. Interestingly, selected hybrids presented a similar range of variation, from a minimum 4.99 g to a maximum of 11.75 g. Of the 212 accessions analysed, 43 accessions presented less than 6 g sugars, 159 accessions presented 6–9 g sugars and only 10 were greater than 9 g. Linamarin was not detected in the genotypes analysed. Applications and perspectives for cassava breeding programmes are discussed. (Résumé d'auteur)