Kluyveromyces marxianusCCT 7735 shows potential for producing ethanol from lactose; however, its low ethanol tolerance is a drawback for its industrial application. The first aim of this study was to obtain four ethanol-tolerantK. marxianusCCT 7735 strains (ETS1, ETS2, ETS3, and ETS4) by adaptive laboratory evolution. The second aim was to select among them the strain that stood out and to evaluate metabolic changes associated with the improved ethanol tolerance in this strain. The ETS4 was selected for displaying a specific growth rate higher than the parental strain under ethanol stress (122%) and specific ethanol production rate (0.26 g/g/h) higher than those presented by the ETS1 (0.22 g/g/h), ETS2 (0.17 g/g/h), and ETS3 (0.17 g/g/h) under non-stress condition. Further analyses were performed with the ETS4 in comparison with its parental strain in order to characterize metabolic changes. Accumulation of valine and metabolites of the citric acid cycle (isocitric acid, citric acid, and cis-aconitic acid) was observed only in the ETS4 subjected to ethanol stress. Their accumulation in this strain may have been important to increase ethanol tolerance. Furthermore, the contents of fatty acid methyl esters and ergosterol were higher in the ETS4 than in the parental strain. These differences likely contributed to enhance ethanol tolerance in the ETS4.