Although asterinaceous fungi have been studied for many years, all previous attempts to isolate, cultivate, and propagate these fungi in vitro have failed. This paper provides the first reports of in vitro isolation of representative strains of species belonging to five fungi from different genera belonging to Asterinales. To confirm if the sequences of DNA obtained from the mycelia are the same obtained in the direct extraction, a phylogenetic analysis of nuc LSU rDNA was performed. This paper reports for the first time the success of in vitro culturing of asterinaceous fungi using the ascospores ejection technique, opening perspectives of studies of genetics, physiology, among other aspects of the biology for this very understudied group of fungi. • Asterinales are phytopathogenic fungi with a worldwide distribution, which absorb nutrients through the haustoria. • HM628777 For Asterinales, cultivation depends on the technique and not on the medium used. • The isolation of Asterinales opens news perspectives with the possibility of genetic studies, physiology, and pathogenicity. [ABSTRACT FROM AUTHOR]