Abstract During the 2013 and 2014 growth seasons, 526 single spore isolates of Botrytis cinerea were collected from naturally infected ‘Thompson Seedless’ table grape flowers from fifteen orchards in three regions of the Central Valley of Chile. The isolates were tested for resistance to azoxystrobin, boscalid, fenhexamid, fludioxonil and pyrimethanil. Among the 526 isolates, 106 (20.15%) were sensitive to all fungicides tested; from north to south, the frequency of sensitive isolates in the regions of Valparaiso, Metropolitana and O’Higgins ranged from 48.15% to 21.1% and 5.88%, respectively. Four hundred and twenty isolates (79%) showed resistance to single or multiple fungicides, 134 (25.4%) were simultaneously resistant to azoxystrobin and pyrimethanil. No fludioxonil-resistant isolates were found, indicating that fludioxonil has great potential for gray mold control in table grapes in Chile. From sixty randomly selected B. cinerea isolates, only the azoxystrobin-resistant isolates carried the G143A point mutation; according to the cytochrome b (cyt b) gene structure, the third intron Bcbi-143/144 was only detected in the azoxystrobin-sensitive isolates. The H272R and H272Y point mutations in the succinate dehydrogenase subunit B (sdhB) gene were associated only with the boscalid-resistant isolates. The F412S and F412V point mutations were found in the sequenced erg27 gene of randomly selected fenhexamid-resistant isolates. These results contribute to the knowledge of B. cinerea fungicide resistance for table grape vine crops in Central Chile, particularly for the development of multiple-resistance and the associated resistance mechanisms of azoxystrobin, boscalid and fenhexamid-resistant isolate populations. Antiresistance strategies are discussed in a general manner.