AbstractThis study focuses on the role of the qualitative leaf wax composition in modulating the cuticular water loss using a Populus × canescens cer6 mutant line, which accumulates C34–C46 wax ester dimers and is reduced in wax monomers >C24. The two literature‐based hypotheses to be tested were the importance of the amount of wax esters and the weighted mean carbon chain length in restricting cuticular water loss. The main results were acquired by chemical analysis of cuticular wax and gravimetric cuticular transpiration measurements. Besides additional physiological measurements, the leaf surface properties were characterised by scanning electron microscopy and spectrophotometric light reflectance quantification. Mutation of the CER6 gene resulted in striking changes in qualitative wax composition but not quantitative wax amount. Based on the strong accumulation of dimeric wax esters, the relative proportion of esters increased to >90%, and the weighted mean carbon chain length increased by >6 carbon atoms. These qualitative alterations were found to increase the cuticular transpiration of leaves by twofold. Our results do not support the hypotheses that enhanced amounts of wax esters or increased weighted mean carbon chain lengths of waxes lead to reduced cuticular transpiration.