ESFY phytoplasma (European stone fruit yellows phytoplasma) is nowadays one of the most important plant diseases, especially on apricots and peaches, and it belongs to the list of organisms for which quarantine is required in the Czech Republic. The aim of this study was to determine the best period for tissue extraction and the best technique for ESFY detection. It was also to investigate the possibility of isolating DNA for use in ESFY detection from the leaf-stalks of randomly chosen symptomatic and asymptomatic apricot trees. Results of the amplification of DNA extracted from leaf-stalk and phloem sampled from 2-year old woody shoots during the years 2003 and 2004 were statistically analysed and compared, and visible disease symptoms were simultaneously evaluated and compared to the results of molecular detection. DNA isolation from leaf-stalks can be considered as less significant and reliable than isolation from phloem sampled from 2-year old woody shoots.