Aims: Investigation of the meiotic segregation of karyotypes and physiological traits in indigenous Saccharomyces strains isolated from Aglianico (South Italy) red wine. Methods and Results: Segregation was studied in F1 and F2 descendants. Tetrads were isolated from sporulating cultures by micromanipulation. The spore clones were subjected to karyotype analysis by pulse-field gel electrophoresis (Bio-Rad model CHEF-DR II) and to various physiological tests. Certain chromosomes of the isolates showed 2 : 2 segregation patterns in F1 but proved to be stable in F2. The ability of cells to utilize maltose also segregated in a 2 : 2 manner in F1 and did not segregate in F2. Resistance to CUSO4, SO2 tolerance, the fermentative power and the production of certain metabolites segregated in both F1 and F2 generations and showed patterns indicating the involvement of polygenic regulation. Conclusions: The analysis revealed a high degree of genetic instability and demonstrated that meiosis can improve chromosomal and genetic stability. Significance and Impact of the Study: Winemaking is critically dependent on the physiological properties and genetic stability of the fermenting Saccharomyces yeasts. Selection of clones from F2 or later generations can be a method of reduction of genetic instability.