Calyx-end rot (CER) is an important disease on apple in Chile, affecting the quantity and quality of fruits. Recently the incidence of CER has been reported between 0.1 to 0.2% to harvest reaching an 2% during storage at 0°C. However, a deep study of the characterization of species of Botrytis obtained from apples with CER has not yet been studied. Therefore, twenty isolates representatives obtained from symptomatic apples were characterized culturally, morphologically, molecularly and pathogenically. Botrytis isolates were classified according to sporulation degree in culture medium potato dextrose agar acidulated (APDA) and pea agar (PA). Shape and size of conidia and conidiophore were evaluated in PA media. Sclerotial production was evaluated on APDA. All isolates were molecularly characterized by amplification of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), DNA-dependent RNA polymerase subunit II (RPB2) and necrosis and ethylene-inducing proteins (NEP1) genes. Pathogenicity tests were carried out on cultivars Cripps Pink, Fuji, Granny Smith, Modi, Premium Gala, Braeburn, Scarlette and Red Chief. Based in cultural, morphological and phylogenetic analysis the isolates were identified as B. cinerea and B. caroliniana. All isolates were pathogenic in the apple cultivars tested, showed difference of virulence between isolates of both Botrytis species. Additionally, this isolates were sensitive in vitro to several fungicides. B. cinerea was the most frequent species of Botrytis causing CER in apple fruits in Maule Region, Chile.