Pine pitch canker (PPC), caused by Fusarium circinatum, is considered among the most important diseases affecting pines in many locations throughout the world. In Europe, F. circinatum is currently present in the Iberian Peninsula, posing a high risk of its spread into currently disease-free countries in Europe. In the present study, the susceptibility of Pinus sylvestris, Picea abies, and Larix decidua originating in the Czech Republic to F. circinatum was tested. Furthermore, the presence of asymptomatic yet infected seedlings was also checked. This study demonstrated the pathogenicity of F. circinatum to the Czech provenance of P. sylvestris, whereas Picea abies and Larix decidua proved to be tolerant. The reisolation of F. circinatum beyond the inoculation point demonstrated that this quarantine pathogen is able to infect the three conifers tested, giving rise to asymptomatic seedlings for at least eight and a half months. To our knowledge, this is the first study in which the presence of symptomless seedlings has been recorded in the genera Picea and Larix. This finding points out that the European legislation would fail to avoid the risk of new introductions via symptomless seedlings, since this legislation is only restricted to plants of the genus Pinus and the species Pseudotsuga menziesii.