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A rapid and efficient analytical method for the determination of glycerol in wines is described. This method utilizes high-performance thin layer chromatography (HPTLC) plates coupled with an automated multiple development system with an elution gradient based on acetonitrile–acetone–hexane on silica gel layers. The absence of clean-up procedures, sometimes only centrifugation, makes this method suitable also for the large-scale control of alcoholic beverages. In particular the capacity of different wine yeast species (Saccharomyces cerevisiae, Zygosaccharomyces bailii, Kloeckera apiculata and Saccharomycodes ludwigii) to produce glycerol was determined. Generally, the strains of S. cerevisiae produced elevated amounts of glycerol together with Z. bailii, whereas K. apiculata strains formed the lowest amounts of glycerol, exhibiting also a great strain variability.