Abstract: Fusarium circinatum is the causal agent of pine pitch canker (PPC), one of the most devastating forest diseases worldwide. This fungus causes severe damping-off in pine seedlings and growth reduction, wilting and the development of cankers in pine forests and plantations. A draft of the complete genome sequence of this phytopathogen was recently made available. This information was used to annotate in silico the gene Fcrho1 as a putative homolog of Rho1 GTPase genes. In this study, we generated Fcrho1 deletion mutants in two F. circinatum wildtype strains isolated from damaged trees in northern Spain. For that, we used a modified version of the OSCAR methodology, an approach not previously used in F. circinatum that allows the generation of deletion constructs in a single cloning step. The conidiation and spore germination of the resulting deletion mutants were not affected, neither the hyphal morphology. However, the mutant strains showed significantly reduced growth in vitro and more foamy macroscopic hyphal morphology than their corresponding ectopic and wildtype strains. Finally, an in vivo virulence assay showed that the reduced in vitro growth rate characteristic to the deletion mutants does not impact their pathogenicity.