Cercospora beticola strains with laboratory induced resistance to tetraconazole were compared with their parental WT sensitive strains to evaluate the effects of resistance on fitness and assess whether any change in the sterol biosynthetic pathway was associated to the reduced fungicide sensitivity. In vitro growth rate on agar media and pathogenicity were found to be negatively affected by resistance. The main functional sterols in C. beticola WT strains under investigation were ergosterol, brassicasterol and ergosta-7,22-dienol. Resistant strains showed the same qualitative sterol composition, ruling it out as, per se, a cause for resistance. On the basis of the sterols detected both in sensitive and resistant strains, a possible biosynthetic pathway to the three functional sterols is proposed. Tetraconazole treatment caused, in all sensitive strains, the immediate accumulation of 14α-methylated sterols, which, for inhibitor concentrations up to EC50 values, were, in order of abundance, 14α-methylergosta-8,24(28)-dien-3β,6α-diol, eburicol and obtusifoliol. However the data do not support a critical role of the 14-methyl-3,6-diol derivative in the growth arrest of C. beticola. As main difference between sensitive and resistant strains, the formers were found to accumulate higher amounts of 14α-methylated sterols. Although the data do not allow to establish a specific mechanism for resistance, some molecular mechanisms such as target site alterations and sterol biosynthetic pathway can be ruled out as a possible cause for reduced sensitivity.