Fusarium subglutinans has been associated with many hosts. Some isolates of the fungus are responsible for pitch canker disease of pines. A South African population of F. subglutinans from pine seedlings was characterized by comparing it to isolates from various hosts, pine and non-pine. A total of 26 isolates representing six different hosts were selected for the study. Cultural and morphological characteristics were studied. Virulence of isolates was tested on 1-yr-old Pinus patula seedlings. Sexual compatibility of isolates of F. subglutinans with B- and E-tester strains, and crossings of pine isolates in all possible combinations, were performed. Pine isolates were also compared with pitch canker and non-pine isolates using random amplified polymorphic DNAs (RAPDs). All isolates were morphologically characteristic of F. subglutinans, but differed somewhat in cultural features. Only isolates from pine were pathogenic to P. patula seedlings. Virtually no isolates were sexually compatible with the fertile tester strains. An exception was isolate MRC 115 from maize. Some pine isolates (MRC 6211 and MRC 6217) were, however, sexually compatible with South African (MRC 6213) and pitch canker (MRC 6229) isolates. Cluster patterns constructed from RAPD profiles revealed that isolates clustered according to host. South African isolates from pine were related to the pitch canker isolates from other areas, and were similar to non-pine isolates only in morphology. Virulence, genetic analyses and composite RAPD data suggest that pine isolates originate from the same gene pool, and probably represent a distinct new biological species.