Banana has been currently indicated as a good source of fructooligosaccharides (FOS), which are considered to be functional components of foods. However, significant differences in their amounts in bananas have been observed in the literature. This work aims to identify and quantify FOS during ripening in different banana cultivars belonging to the most common genomic groups cultivated in Brazil. Considering that these differences can be due to cultivar, stage of ripening, and the methodologies used for FOS analyses, sugar contents were analyzed by high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and gas chromatography-mass spectrometry (GC-MS). An initial screening of eight cultivars (Ouro, Nanicao, Prata, Maca, Mysore, Pacovan, Terra, and Figo) in a full-ripe stage showed that 1-kestose, the first member of the FOS series (amounts between 297 and 1600 mu g/g of DM), was accumulated in all of them. Nystose, the second member, was detected only in Prata cultivar. Five of the cultivars were analyzed during ripening, and a strong correlation could be established with a specific sucrose level (similar to 200 mg/g of DM), which seems to trigger the synthesis of 1-kestose (the low amounts of FOS, below the functional recommended dose, indicates that banana cannot be considered a good source of FOS).