Root rot of avocado (Persea americana) is an important disease in seedling nurseries as well as in the field in eastern and southern Australia. During an investigation into the causal organisms of avocado root rot, 19 isolates of Gliocladiopsis were obtained from necrotic lesions on avocado roots and examined by morphology and comparison of DNA sequences from three gene loci (the internal transcribed spacer region of the nuclear rDNA, Histone H3 and β-tubulin). Three new species of Gliocladiopsis are described as a result of phylogenetic analysis of these data. One of the new species, G. peggii, formed a monophyletic group that may represent an unresolved species complex as it contained a polytomy that included a well-supported clade comprising two subclades. Gliocladiopsis peggii is sister to G. mexicana, which is known from soil in Mexico. The remaining two new species, G. whileyi and G. forsbergii, formed a clade sister to G. curvata, which is known from Ecuador, Indonesia and New Zealand.