Fusarium circinatum is the causal agent of the pine disease commonly referred to as pitch canker. During 2001, a Fusarium species was isolated from dying Pinus radiata clonal hedges in various forestry nurseries in Chile and was subsequently identified as F. circinatum. The aim of the study reported here was to provide a detailed characterization of Chilean isolates of the fungus. Morphological characters included microconidia carried on false heads and produced on polyphialides. Sterile coils and conidiophores on erect aerial mycelium were evident on synthetic, low nutrient agar. Furthermore, perithecia exuding viable ascospores were produced when isolates were crossed in all possible combinations with the mating tester strains representing the H mating population of Gibberella fujikuroi species complex. PCR-RFLP analysis of the histone H3 gene region, routinely used to distinguish between members of the G. fujikuroi complex, further confirmed the identification of the isolates as F. circinatum. DNA sequence data obtained for the same gene region placed the isolates within a well-characterized G. circinata clade. These studies provide unequivocal evidence that the pitch canker pathogen is well established on pines in Chilean nurseries.