The absolute quantities and the stereoisomeric ratios of R, S-2-hydroxybutanone and R, R-, S, S-, R, S ( meso)-2,3-butanediol were determined in batteries of traditional balsamic vinegar (TBV) by gas chromatography-mass spectrometry (GC-MS), using a chiral capillary column, to evaluate if such parameters could be used to differentiate TBV differently aged and from different producers. Results showed that the initial amounts of 2-hydroxybutanone and 2,3-butanediol were quite variable, as a function of the producer of the vinegar; moreover, the 2-hydroxybutanone amount decreased during aging while 2,3-butanediol increased. Initially, the R-2-hydroxybutanone form prevails, and then the R/ S ratio decreased regularly during aging with some exceptions attributed to the addition of new barrels during the battery management. With regard to the 2,3-butanediol isomers, the most abundant was the R, R form, slowly transformed into the R, S and S, S isomers during aging. The GC-MS method used is easy and fast and could allow for a quick control of the maturation level of the vinegar.