Flor yeast velum is a biofilm formed by certain yeast strains that distinguishes biologically aged wines such as Sherry wine from southern Spain from others. Although is the most common species, 5.8 S-internal transcribed spacer (ITS) restriction fragment length polymorphism analyses have revealed the existence of non- species. In order to uncover the flor microbiota diversity at a species level, we used ITS (internal transcribed spacer 1)-metabarcoding and matrix-assisted laser desorption/Ionization time of flight mass spectrometry techniques. Further, to enhance identification effectiveness, we performed an additional incubation stage in 1:1 wine:yeast extract peptone dextrose (YPD) before identification. Six species were identified: , , , , , and , two of which were discovered for the first time ( and ) in Sherry wines. We analyzed wines where non- yeasts were present or absent to see any potential link between the microbiota and the chemical profile. Only 2 significant volatile chemicals (out of 13 quantified), ethanol and ethyl lactate, and 2 enological parameters (out of 6 quantified), such as pH and titratable acidity, were found to differ in long-aged wines. Although results show a low impact where the non- yeasts are present, these yeasts isolated from harsh environments (high ethanol and low nutrient availability) could have a potential industrial interest in fields such as food microbiology and biofuel production.