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 RIS 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,Sand 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.