This study evaluated the genetic variability and in vitro susceptibility patterns of isolates of Neoscytalidium dimidiatum and Scytalidium hyalinum from different geographical origins. Partial sequences of four loci (the ITS region and D1/D2 domains of the 28S rRNA gene and the tubulin and chitin synthase genes) were analysed. Among a total of 1970 bp sequenced in 24 isolates, 7 polymorphic positions (0.36%) were detected, representing five different sequence types (ST1-ST5), from which two (ST2 and ST3) were detected exclusively in isolates from plants, two (ST1 and ST5) were found only in clinical isolates and one (ST4) was observed in isolates from humans and from a mango tree. We propose subordinating S. hyalinum as a variety of N. dimidiatum. Amphotericin B was the most active drug, but low minimum inhibitory concentrations were also detected for voriconazole, terbinafine and anidulafungin.