The aim of the present study was to evaluate the effect of post-flowering chilling of sweet cherry (Prunus avium L.) on the content of biochemical parameters in the leaf (chloroplast pigments, sugars and phenolics). The effect of chilling was investigated in two experiments. Potted 2-year-old trees of cv. 'Grace Star' and 'Schneiders' were exposed to one, two or three consecutive overnight chillings at an average air temperature of 4.7 °C (Experiment I), but in the following year only trees of 'Grace Star' were chilled at 2.2 °C (Experiment II), 3 to 7 weeks after flowering. The analysis of the biochemical parameters was performed by high performance liquid chromatography combined with electrospray ionization mass spectrometry. Chilling at 4.7 °C caused little or no stress, while 2.2 °C induced more intense stress with increased zeaxanthin, sugar and phenolic content in leaves, while exposure of trees to higher temperatures and closer to flowering showed no changes. Two or three consecutive overnight chilling periods increased the phenolic content and enhanced the accumulation of zeaxanthin in the leaves. Sucrose, sorbitol, fructose, total sugar, and total flavonoid content in leaves increased within 48 h after chilling. Zeaxanthin epoxidized within 24 h after one and 48 h after one and two consecutive overnight chillings.